Alternative splicing factors polynucleotides, polypeptides and uses thereof

ABSTRACT

The invention provides isolated polynucleotides and their encoded proteins that are involved in splicing or modulating splicing activity. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering splicing protein content and/or composition of plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application which claims priority toU.S. Ser. No.10/956,852 filed Oct. 1, 2004 which is a utilityapplication which claims priority to U.S. Ser. No. 60/509,551 filed Oct.8, 2003 and to U.S. Ser. No. 60/557,370 filed Mar. 29, 2004 thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to plant molecular biology. Morespecifically, it relates to nucleic acids and methods for modulatingtheir expression in plants in order to modulate gene regulation and toselectively express polypeptides.

BACKGROUND OF THE INVENTION

A complex network of regulatory pathways control gene expression ineukaryotes. Environmental constraints, such as nutrient availability,mitogenic signals such as growth factors or hormones, as well asdevelopmental cues such as the transition from vegetative toreproductive, modulate transcription and translation in plants. Geneswhich are involved in this modulation can be introduced into a plant andused to affect phenotype in adventitious or desirable ways.

The development of methods for the introduction of foreign genes intoorganisms has had a profound impact on fields of medicine andagriculture and has further expanded understanding of regulatorymechanisms involved in gene expression. While the movement of geneswithin plant species or between closely related plant species bytraditional methods based on sexual reproduction has played an importantrole in crop improvement for most of this century, the pace of cropimprovement by such methods has been slow and limiting due to thereliance on naturally occurring genes. Recent advances in the field ofgenetic engineering have led to the development of genetictransformation methods that allow the introduction of recombinant DNAinto organisms. The recombinant DNA methods which have been developedhave greatly extended the sources from which genetic information can beobtained for crop improvement. Recently, new crop plant varieties,developed through recombinant DNA methods, have reached the marketplace.Genetically engineered soybeans, maize, canola, cotton and other cropsare now widely utilized by North America farmers.

Genes from plants and other entirely unrelated organisms are beingcloned, genetic regulatory signals are being deciphered, and genesconferring new traits, are being transferred. Introduction of singlegenes or of combinations of genes through stacking pose challenges incontrolling the expression of the transferred genes. Many of the recentadvances in plant science have resulted from application of theanalytical power of recombinant DNA technology coupled with planttransformation and an understanding of gene regulatory processes. Theseapproaches facilitate studies of the effects of specific genealterations and additions on plant development and physiology and makepossible the direct manipulation of genes to bio-engineer improved plantvarieties.

While some success has already been achieved in improving crop plantsthrough the introduction and regulation of recombinant DNA, the progressof genetic engineers working to improve many important crop species isimpeded by inefficient methods and limited choices of gene regulation incrop plants. Thus, improved methods for controlling gene expression inplants and the transformed plants regenerated there from are desired.

SUMMARY OF THE INVENTION

Methods and compositions are provided for producing transformed plant,cells, plants, plant embryos and increasing plant transformationefficiency. The methods involve transforming a plant cell with asplicing factor polynucleotide or introducing a splicing factorpolypeptide or RNA. Expression of the splicing factor may be transient,stable or inducible. Levels may be modulated to modulate geneexpression, increase transformation efficiency or to modulate splicingto modulate phenotype; especially phenotypes associated with agronomictraits. The methods and compositions of the invention find use inagriculture, particularly in modulating gene expression in crop plants,and in transforming crop plants that display low transformationefficiencies with existing transformation methods. The methods andcompositions of the invention are also useful in providing selectiveexpression of polypeptides through alternative splicing mechanisms. Alsoprovided are transgenic plants and seeds thereof.

DETAILED DESCRIPTION OF THE INVENTION SEQUENCES

-   Seq. ID no. 1 is the DNA sequence of zmSRp30 (ATG start bp40, TGA    stop bp-820).-   Seq. ID no. 2 is the polypeptide sequence of zmSRp30.-   Seq. ID no. 3 is the DNA sequence of zmSRp30′(ATG start bp40, TGA    stop bp-763).-   Seq. ID no. 4 is the polypeptide sequence of zmSRp30′.-   Seq. ID no. 5 is the DNA sequence of zmSRp31 (ATG start bp-82, TGA    stop bp-910).-   Seq. ID no. 6 is the polypeptide sequence of zmSRp31.-   Seq. ID no. 7 is the DNA sequence of zmSRp31′(ATG start bp-82, TAA    stop bp-841).-   Seq. ID no. 8 is the polypeptide sequence of zmSRp31′.-   Seq. ID no. 9 is the DNA sequence of zmSRp32(ATG start bp-104, TGA    stop bp-955).-   Seq. ID no. 10 is the polypeptide sequence of zmSRp32.-   Seq. ID no. 11 is the DNA sequence of zmSRp32′(ATG start bp-104, TGA    stop bp-875).-   Seq. ID no. 12 is the polypeptide sequence of zmSRp32′.-   Seq. ID no. 13 is the DNA sequence of zmSRp32″(ATG start bp-104, TAG    stop bp-554).-   Seq. ID no. 14 is the polypeptide sequence of zmSRp32″.-   Seq. ID no. 28 the DNA sequence of the genomic zmSRp31.-   Seq. ID no. 29 the DNA sequence of the genomic zmSRp32.-   Seq. ID no. 30 the DNA sequence of the genomic zmSRp30.

DEFINITIONS

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, rolling circle, ligase chain reaction (LCR) system, nucleic acidsequence based amplification (NASBA, Cangene, Mississauga, Ontario),Q-Beta Replicase systems, transcription-based amplification system(TAS), and strand displacement amplification (SDA). See,. e.g.,Diagnostic Molecular Microbiology: Principles and Applications, D. H.Persing et al., Ed., American Society for Microbiology, Washington, D.C.(1993). The product of amplification is termed an amplicon.

As used herein, “antisense orientation” includes reference to a duplexpolynucleotide sequence that is operably linked to a promoter in anorientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

As used herewithin, “cereal grasses” includes wheat, oat and corn.

As used herein, “chromosomal region” includes reference to a length ofchromosome that can be measured by reference to the linear segment ofDNA that it comprises. The chromosomal region can be defined byreference to two unique DNA sequences, i.e., markers.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide of the present invention isimplicit in each described polypeptide sequence and incorporated hereinby reference.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity, or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe native protein for it's native substrate. Conservative substitutiontables providing functionally similar amino acids are well known in theart.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Serine (S), Threonine (T);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).-   See also, Creighton (1984) Proteins W.H. Freeman and Company.

By “encoding” or “encoded”, with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal, and fungalmitochondria, the bacterium Mycoplasma capricolum (Proc. Natl. Acad.Sci. U.S.A. 82:2306-2309 (1985)), or the ciliate Macronucleus, may beused when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al., Nucl. Acids Res. 17:477498 (1989)).Thus, the maize preferred codon for a particular amino acid can bederived from known gene sequences from maize. Maize codon usage for 28genes from maize plants are listed in Table 4 of Murray et al., supra.

As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (non-synthetic), endogenous, catalytically activeform of the specified protein. A full-length sequence can be determinedby size comparison relative to a control that is a native(non-synthetic) endogenous cellular form of the specified nucleic acidor protein. Methods to determine whether a sequence is full-length arewell known in the art including such exemplary techniques as northern orwestern blots, primer extension, S1 protection, and ribonucleaseprotection. See, e.g., Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to knownfull-length homologous (orthologous and/or paralogous) sequences canalso be used to identify full-length sequences of the present invention.Additionally, consensus sequences typically present at the 5′ and 3′untranslated regions of mRNA aid in the identification of apolynucleotide as full-length. For example, the consensus sequenceANNNNAUGG, where the underlined codon represents the N-terminalmethionine, aids in determining whether the polynucleotide has acomplete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition, regulatory signals, neighboring sequence, and/or genomiclocus by deliberate human intervention. For example, a promoter operablylinked to a heterologous structural gene is from a species differentfrom that from which the structural gene was derived, or, if from thesame species, one or both are substantially modified from their originalform. A heterologous protein may originate from a foreign species or, iffrom the same species, is substantially modified from its original formby deliberate human intervention.

By “host cell” is meant a cell that contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such. as E. coli, or eukaryotic cells such asyeast, insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledenous plant cells. One monocotyledonoushost cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). “Introduced” may alsorefer to crossing one plant containing a nucleic acid to another plantso as to incorporate the nucleic acid in their progeny.

The term “isolated” refers to material, such as a nucleic acid or aprotein, which is: (1) substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment; or (2)if the material is in its natural environment, the material has beensynthetically (non-naturally) altered by deliberate human interventionto a composition and/or placed at a locus in the cell (e.g., genome orsubcellular organelle) not native to a material found in thatenvironment. The alteration to yield the synthetic material can beperformed on the material within or removed from its natural state. Forexample, a naturally occurring nucleic acid becomes an isolated nucleicacid if it is altered, or if it is transcribed from DNA that has beenaltered, by non-natural, synthetic (i.e., “man-made”) methods performedwithin the cell from which it originates. See, e.g., Compounds andMethods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S.Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in EukaryoticCells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurringnucleic acid (e.g., a promoter) becomes isolated if it is introduced bynon-naturally occurring means to a locus of the genome not native tothat nucleic acid. Nucleic acids that are “isolated” as defined herein,are also referred to as “heterologous” nucleic acids.

As used herein, “splicing factor nucleic acid” means a nucleic acidcomprising a polynucleotide “splicing factor polynucleotide” encoding asplicing factor polypeptide. “As used herein, “recalcitrant” meanshaving a low level of transformation efficiency with few or notransgenic events per unit time and resources. Thus, a plant that isrecalcitrant to transformation, in the current art, has a transformationefficiency that is less than GS3. For example, low rates ofregeneration, embryogenesis and/or increased susceptibility to increasedsusceptibility to cell damage may be factors leading to exhibitingrecalcitrant characteristics.

As used herein, “localized within the chromosomal region defined by andincluding” with respect to particular markers includes reference to acontiguous length of a chromosome delimited by and including the statedmarkers.

As used herein, “marker” includes reference to a locus on a chromosomethat serves to identify a unique position on the chromosome. A“polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes in that pair to be followed. A genotype may be defined byuse of one or a plurality of markers. A “selectable marker” refers to atrait that aids in the identification of desired characteristics. Forexample, the “selectable marker” bar confers bialaphos resistance uponthe acquiring organism allowing identification through growth on thenormally toxic bialaphos.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules that comprise and substantially represent the entire genome ofa specified organism. Construction of exemplary nucleic acid libraries,such as genomic and cDNA libraries, is taught in standard molecularbiology references such as Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in MolecularBiology, F. M. Ausubel et al., Eds. Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.(1994 Supplement).

As used herein “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A plant of interest is Zea mays.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof, thathave the essential nature of a natural ribonucleotide in that theyhybridize to nucleic acids in a manner similar to naturally occurringnucleotides. A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Exemplary modifications aredescribed in most basic texts, such as, Proteins—Structure and MolecularProperties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas, for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pp.1-12 in PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) andRattan et al., Protein Synthesis: Posttranslational Modifications andAging, Ann. N.Y., Acad. Sci. 663:48-62 (1992). It will be appreciated,as is well known and as noted above, that polypeptides are not alwaysentirely linear. For instance, polypeptides may be branched as a resultof ubiquitination, and they may be circular, with or without branching,generally as a result of post translation events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Modifications can occur anywherein a polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termin. In fact, blockage of theamino or carboxyl group in a polypeptide, or both, by a covalentmodification, is common in naturally occurring and syntheticpolypeptides and such modifications may be present in polypeptides ofthe present invention, as well. For instance, the amino terminal residueof polypeptides made in E. coli or other cells, prior to proteolyticprocessing, almost invariably will be N-formylmethionine. Duringpost-translational modification of the peptide, a methionine residue atthe NH₂-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription that is involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue-preferred”. A “cell type”promoter primarily drives expression in certain cell types in one ormore organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter that is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-preferred, cell type, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.

The term “cell cycle polypeptide” refers to one or more amino acidsequences, in glycosylated or non-glycosylated form, involved in theregulation of cell division. The term is also inclusive of fragments,variants, homologs, alleles or precursors (e.g., preproproteins orproproteins) thereof.

The term “splicing factor polypeptide” or “SF” refers to one or moresplicing factor amino acid sequences involved in the regulation ofsplicing. The term “SR polypeptide” or “SR” refers to one or more aminoacid sequences which are RNA-binding proteins containing repeatingarginine and serine residues (SR proteins) and are implicated inconstitutive and/or alternative splicing of pre-mRNA. SR proteins areinvolved in the selection and utilization of splice sites byspliceosomes; SF2/ASF-like polypeptides are a subset of SR proteins. Anamino acid motif comprising “SWQDLKD” (SEQ ID NO: 31) is characteristicof SF2/ASF-like polypeptides.

These terms are also inclusive of fragments, variants, splicingalternatives, homologs, alleles, glycosylated or precursors (e.g.,preproproteins or proproteins) thereof.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, preferably 90% sequenceidentity, and most preferably 100% sequence identity (i.e.,complementary) with each other.

The term “specifically reactive” includes reference to a bindingreaction between an antibody and a protein having an epitope recognizedby the antigen binding site of the antibody. This binding reaction isdeterminative of the presence of a protein having the recognized epitopeamongst the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to an analyte having the recognized epitope to asubstantially greater degree (e.g., at least 2-fold over background)than to substantially all other analytes lacking the epitope which arepresent in the sample.

The terms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Generallyhybridization is conducted for a time in the range of from four tosixteen hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-lnterscience, New York (1995).

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

As used herein “stable transformation” refers to the transfer of anucleic acid fragment into a genome of a host organism (this includesboth nuclear and organelle genomes) resulting in genetically stableinheritance. In addition to traditional methods, stable transformationincludes the alteration of gene expression by any means includingchimerplasty or transposon insertion.

As used herein “Transient Transformation” refers to the transfer of anucleic acid fragment into the nucleus (or DNA-containing organelle) ofa host organism resulting in gene expression without integration andstable inheritance.

As used herein “Modified cells” are cells that have been transformed.

As used herein “Re-transformation” refers to the transformation of amodified cell.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

As used herein, “vivipary” refers to failure of the embryo to enterdevelopmental arrest, causing precocious germination of the seed on themother plant (McCarty et al., 1989).

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence a gap penalty is typically introduced andis subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG® programs,Accelrys, Inc., San Diego, Calif.).; the CLUSTAL program is welldescribed by Higgins and Sharp, Gene 73:237-244 (1988); Higgins andSharp, CABIOS 5:151-153 (1989); Corpet et al., Nucleic Acids Research16:10881-90 (1988); Huang et al., Computer Applications in theBiosciences 8:155-65 (1992), and Pearson et al., Methods in MolecularBiology 24:307-331 (1994). The BLAST family of programs which can beused for database similarity searches includes: BLASTN for nucleotidequery sequences against nucleotide database sequences; BLASTX fornucleotide query sequences against protein database sequences; BLASTPfor protein query sequences against protein database sequences; TBLASTNfor protein query sequences against nucleotide database sequences; andTBLASTX for nucleotide query sequences against nucleotide databasesequences. See, Current Protocols in Molecular Biology, Chapter 19,Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York(1995).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences that may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem. 17:149-163 (1993)) and XNU (Claverie andStates, Comput. Chem. 17:191-201 (1993)) low-complexity filters can beemployed alone or in combination.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences includes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g. chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions have “sequence similarity” or “similarity”.Means for making this adjustment are well known to those of skill in theart. Typically this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, e.g.,according to the algorithm of Meyers and Miller, Computer Applic. Biol.Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. For purposes of defining the invention, % identity onthe nucleic acid level is determined by the BESTFIT DNA SequenceAlignment software on Genescape using a gap weight of 50 and a lengthweight of 3. For purposes of defining the invention, % identity on theamino acid level is determined by the BESTFIT DNA Sequence Alignmentsoftware on Genescape using a gap weight of 12 and a length weight of 4.

(e) (i) The term “substantial identity” of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or highersequence identity to, compared to a reference sequence using one of thealignment programs described using standard parameters. One of skillwill recognize that these values can be appropriately adjusted todetermine corresponding identity of proteins encoded by two nucleotidesequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning and the like. Substantial identityof amino acid sequences for these purposes normally means sequenceidentity of at least 85%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.However, nucleic acids which do not hybridize to each other understringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

(e) (ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the reference sequence over a specified comparison window.Optimal alignment can be conducted using the homology alignmentalgorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). Peptideswhich are “substantially similar” share sequences as noted above exceptthat residue positions which are not identical may differ byconservative amino acid changes.

The present invention provides novel methods of using SR polypeptidesand polynucleotides. Included are methods for increasing transformationfrequencies, increasing crop yield, providing a positive growthadvantage, modulating cell division, transiently modulating celldivision, and for providing a means of identifying transformants.

The present invention provides novel nucleic acids encoding polypeptideshaving RNA binding activity. It further provides novel nucleic acidsencoding polypeptides having RNA splicing activity.

The present invention provides novel nucleic acids encoding SF2/ASF-likegenes zmSRp30, zmSRp31, and zmSRp32, and nucleic acids havingsubstantial identity thereto. The present invention also providespolynucleotides complementary to zmSRp30, zmSRp31 and zmSRp32 andnucleic acids having substantial identity thereto.

The present invention further provides novel nucleic acids encodingSF2/ASF-like genes, zmSRp30, zmSRp31 and zmSRp32, and nucleic acids thathybridize thereto under stringent conditions.

Further provided are exonic and intronic sequences, promoters anduntranslated sequence. Also provided are vectors, expression cassettes,transgenic host cells, transgenic plants, cells and transgenic seedcomprising the polynucleotides and polypeptides of the invention. Thepresent invention provides for methods of growing the plant cell toproduce a regenerated, stably transformed plant. These plants and plantcells include corn, soybean, sorghum, sunflower, safflower, wheat, rice,alfalfa and oil-seed Brassica.

The present invention provides novel methods of affecting the splicingof a virus. It also provides novel methods of affecting the splicing ofGeminiviral transcripts. The present invention further provides novelmethods of affecting the splicing of Rep A. The present inventionfurther provides novel methods of affecting the splicing of a virus suchas wheat dwarf and maize streak virus. The present invention providesnovel methods of increasing resistance to Geminivirus infection (Davieset al., “The Structure, Expression, Functions and Possible Exploitationof Geminivirus Genomes”, Plant DNA Infectious Agents/edited by T. Hohnand J.

Schell, Wien:Springer-Verlag 2:31-52 (1987).

The present invention provides novel polypeptides having RNA bindingactivity. It further provides novel polypeptides having RNA splicingactivity. The present invention provides proteins that may involvemodulation of gene expression in transgenic plants by a trans-splicingmechanism resulting in synthesis of different gene products from thesame pre-mRNA transcripts.

This invention also provides proteins that may stimulate embryogenesisin recalcitrant plant material in order to increase the overallefficiency of transformation process.

The present invention provides novel methods of increasingtransformation efficiency in a plant cell. A responsive target plantcell may be stably transformed with at least one SF2/ASF-like gene in avector to produce a modified target cell. The modified target cell maybe grown under conditions to produce at least one cell division toproduce a progeny cell expressing the SF2/ASF-like polypeptide and then,optionally, the progeny cell is transformed with one or more vectorscontaining a polynucleotide of interest operably linked to a promoter.Also, a responsive target plant cell may be stably transformed with atleast one SF2/ASF-like gene in a vector and a gene of interest toproduce a modified target cell. The gene of interest may be in the samevector or in a separate vector. Alternatively, SF2/ASF-like protein orRNA may be introduced so as to increase transformation efficiency.

The present invention further provides novel methods of modulatingsplicing. The present invention provides novel methods for modulatinggene expression, through modulation of splicing in plants. Methods formodulating SF2/ASF-like splicing proteins in response to environmental,developmental and other stimulus are provided.

In another aspect the invention provides a method for transientlymodulating gene regulation of target cells comprising introducing intothe target cells an isolated splicing polynucleotide, such as a ZmSRpolynucleotide, in sense or antisense orientation operably linked to apromoter driving expression in the target cells, an isolated ZmSRpolypeptide, or an antibody directed against a ZmSR polypeptide. ZmSRpolynucleotides are envisioned to include ZmSRp30, 30′, 31, 31′, 32, 32′and 32″.

In another aspect the invention provides a method for providing a meansof identifying transformants comprising (a) introducing into a targetcell an isolated polynucleotide operably linked to a promoter drivingexpression in the target cell or an isolated SF2/ASF-like polypeptideand (b) selecting for cells exhibiting positive growth advantage.

Further provided are methods of modulating splicing in a plant cellthrough introducing into a plant cell a SF2/ASF-like polynucleotideoperably linked to a promoter to produce a transformed cell and growingthe transformed cell to modulate splicing in the transformed cellcompared to a corresponding non-transformed cell. The promoter may be aninducible, tissue-preferred promoter, an ear or tassel-preferredpromoter.

The present invention provides for methods of modulating splicing in aplant cell wherein transgenes are spliced, the cell cycle is stimulated,or sex determination is affected. Further provided are methods wherethere is modulation of reproductive organ growth, vegetative plantgrowth, yield, apomixis, or flowering time.

The present invention provides for methods of modulating splicing in aplant cell increasing male sterility, abiotic stress tolerance,metabolic activity, photosynthesis, vegetative yield, pathogenresistance, or embryogenesis.

The present invention provides for methods of modulating splicing in aplant cell decreasing vivipary.

The present invention provides novel methods of identifyingtransformants by introducing into a cell a SF2/ASF-like polynucleotideoperably linked to a promoter to produce a transformed cell and growingthe transformed cell so as to increase embryogenesis in the transformedcell and provide a means of identifying transformants.

In addition to the positive influence of transient increases intransformation efficiency, stable expression would be a benefit foridentifying transformants such as in positive selection schemes in therecovery of transgenic plants and plant cells. In a population of cellsand/or callus growing in vitro, cells expressing an SF2/ASF-like gene,such as zmSRp30, zmSRp31 or zmSRp32, will provide differential growthadvantage based simply on their accelerated embryogenesis. It would beexpected that these transgenic cells or cell/clusters would grow morerapidly than their non-transformed counterparts in culture, permittingready identification of transformants. Such a positive growth advantage(imparted by expression of a gene such as zmSRp30, zmSRp31 and zmSRp32),would also be beneficial in other types of transformation strategies,including as examples, protoplast transformation, leaf basetransformation and transformation of cells in meristems. Such growthstimulation may also extend transformation protocols to tissues normallynot amenable to culture.

The present invention provides, inter alia, compositions and methods formodulating (i.e., increasing or decreasing) the total levels of proteinsof the present invention and/or altering their ratios in plants. Thus,the present invention provides utility in such exemplary applications asthe regulation of gene expression, embryogenesis and differentiationthrough splicing. The polypeptides of the present invention can beexpressed at times or in quantities that are not characteristic ofnon-recombinant plants or in recombinant plants without such regulation.

In particular, modulating splicing proteins is expected to provide apositive organ growth advantage and increase crop yield oralternatively, provide a negative growth advantage for use in, forexample, a male sterility system.

The present invention also provides isolated nucleic acid comprisingpolynucleotides of sufficient length and complementarity to a splicinggene to use as probes or amplification primers in the detection,quantitation, or isolation of gene transcripts. For example, isolatednucleic acids of the present invention can be used as probes indetecting deficiencies in the level of mRNA in screenings for desiredtransgenic plants, for detecting mutations in the gene (e.g.,substitutions, deletions, or additions), for monitoring upregulation ofexpression or changes in enzyme activity in screening assays ofcompounds, for detection of any number of allelic variants(polymorphisms) of the gene, or for use as molecular markers in plantbreeding programs. The isolated nucleic acids of the present inventioncan also be used for recombinant expression of splicing polypeptides, orfor use as immunogens in the preparation and/or screening of antibodies.The isolated nucleic acids of the present invention can also be employedfor use in sense or antisense suppression of one or more splicing genesin a host cell, tissue, or plant. Attachment of chemical agents thatbind, intercalate, cleave and/or crosslink to the isolated nucleic acidsof the present invention can also be used to modulate transcription ortranslation. Further, using a primer specific to an insertion sequence(e.g., transposon) and a primer which specifically hybridizes to anisolated nucleic acid of the present invention, one can use nucleic acidamplification to identify insertion sequence inactivated splicing genesfrom a cDNA library prepared from insertion sequence mutagenized plants.Progeny seed from the plants comprising the desired inactivated gene canbe grown to a plant to study the phenotypic changes characteristic ofthat inactivation. See, Tools to Determine the Function of Genes, 1995Proceedings of the Fiftieth Annual Corn and Sorghum Industry ResearchConference, American Seed Trade Association, Washington, D.C., 1995.

Additionally, non-translated 5′ or 3′ regions of the polynucleotides ofthe present invention can be used to modulate turnover of heterologousmRNAs and/or protein synthesis. Further, the codon preferencecharacteristic of the polynucleotides of the present invention can beemployed in heterologous sequences, or altered in homologous orheterologous sequences, to modulate translational level and/or rates.

The present invention also provides isolated proteins comprisingpolypeptides including an amino acid sequence from the splicingpolypeptides as disclosed herein. The present invention also providesproteins comprising at least one epitope from a splicing polypeptide.The proteins of the present invention can be employed in assays forenzyme agonists or antagonists of enzyme function, or for use asimmunogens or antigens to obtain antibodies specifically immunoreactivewith a protein of the present invention. Such antibodies can be used inassays for expression levels, for identifying and/or isolating nucleicacids of the present invention from expression libraries, or forpurification of splicing polypeptides.

Methods are provided to express an SF2/ASF-like gene in atissue-preferred manner. It is an object of this invention to affect eardevelopment to increase yield. It is also an object of this invention toaffect embryo development to increase embryo size, viability,transformability and performance. It is also an object of this inventionto affect tassel development through modulation of SF2/ASF-like gene.Increasing tassel performance may improve pollen count, shed, viabilityor other fertility factors while decreasing tassel performance may leadto male sterility and provide another method of hybrid production.

This invention provides for a novel method of regulation of geneexpression where expression is modulated at the RNA level. A foreigngene is introduced in plant cells by standard transformation methods. Astrong, constitutive promoter such as the maize ubiquitin promoter formonocot plants and the 35SCaMV promoter for expression in dicots controlthe gene expression. The gene structure is modified to contain anintron, or other sequence, with mutated 5′ and/or 3′ splicingrecognitions sites. These splicing sites are not optimally spliced bythe cellular RNA splicing pathways, unless the SF2/ASF-like splicingfactors, including those described in this application, are provided.The intron contains at least one stop codon, in frame with the precedingexonic coding sequences. The pre-mRNA molecules, although produced athigh concentration, are not processed correctly leading to the synthesisof truncated, non-functional protein product. Therefore, the phenotypeexpected from the expressed transgene is not exhibited in thesetransgenic events. The alternative splicing factors, as exemplified bythis application, can be introduced or co-introduced into those plantsby genetic re-transformation, by sexual crossing or by introducing RNAor protein into the cell. The intron sequences will be removed from thepre-mRNA molecules in the re-transformed material, in plant cellswherein the polypeptide is present, or in the progeny of the sexualcross. Functional mRNA molecules will emerge that will be translatedinto a fully functional gene product.

SF2/ASF-Like Genes

Regulation of foreign gene expression in transgenic plants is almostexclusively exercised at the level of transcription. A large number ofpromoter sequences have been isolated and characterized to provide avariety of options for the constitutive, inducible, temporal, or spatialregulation of gene expression. Such approach shares one basicfeature—the expression of a foreign gene activated by the synthesis ofRNA. In many instances, it has been difficult to obtain control ofexpression. For example, steroid inducible systems offer no backgroundactivity in the absence of an inducing factor, but relatively moderateexpression level after induction. On the other hand, heat shockinducible promoters provide a strong activation at elevatedtemperatures, but they are quite “leaky” under normal physiologicalconditions.

RNA-binding proteins containing repeating arginine and serine residues(SR proteins) are implicated in constitutive and alternative splicing ofpre-mRNA. A single pre-mRNA can be processed by alternative splicing toproduce protein isoforms with different physiological functions. In oneextreme case, it has been reported that over 30,000 alternativelyspliced products of just one gene (the Dscam gene) can be expressed inDrosophila (Celofto, A. M. et al., Genetics (2001) 159:599-608). Theprocess contributes to a diversity of gene products and regulation ofgene expression as exemplified by sex determination in Drosophila(Lopez, A. J. et al., Annu. Rev. Genet. (1998) 32:279-305). Pre-mRNAsplicing requires a number of factors organized into a functional entityof the spliceosome. The SR proteins are involved in the selection andutilization of splice sites by spliceosomes, most likely by interactionswith exonic and intronic enhancer sequences (Hastings, M. L. et al.,Curr. Opin. Cell Biol. (2001) 13:302-309).

Alternative splicing has been documented for numerous plant genes.Transcription factors regulating diverse biochemical pathways in plantsare frequently found to produce alternatively spliced products such astranscripts of Vp-1 in wheat (McKibbin, R. S. et al., Proc. Natl. Acad.Sci. USA (2002) 99:10203-10208) (McKibbin et al., 2002), r1 gene inmaize (Procissi et al, 2002), barley and maize MADS-box genes (Montag etal., 1995; Schmitz et al., 2000). The pathogen defense systems oftobacco, Arabidopsis, tomato, and flax rely on alternatively splicedToll-like receptors for signaling pathogen-elicited response (Jordan etal., 2002). The systemic wound response pathway of tomato plantsinvolves alternative splicing of prosystemin (Howe, 2001). The responseto environmental factors such as light or salt concentration as well asthe control of flowering time require SR-like splicing proteins oralternatively spliced gene products (Forment, J. et al., Plant J. (2002)30:511-519); (Macknight, R. et al., Plant Cell (2002) 14:877-888);(Mano, S. et al., Plant J. (1999) 17:309-320).

Alternative splicing is also found in other splicing factor genes,including the polynucleotides of the invention. ZmSRp31 has a fulllength and truncated form and ZmSRp32 has at least three forms: two ofwhich are truncated. ZmSRp30, zmSRp31, and zmSRp32 genes are the firstmaize SF2/ASF-like genes reported.

ZmSRp30, 31, and 32

The maize SF2/ASF-like RNA splicing factor genes, zmSRp30, zmSRp31 andzmSRp32 each have 2 RNA binding domains and one serine-arginine richdomain. The SF2/ASF-like genes have demonstrated splicing activity andthe three polypeptide sequences are 66%-72% identical (77-81% conserved)to each other, Table 1. The first RNA binding domains (amino acids 8-94)of the three polypeptide sequences have 75-76 identical amino acids and81-82 conserved amino acids out of the 87 amino acids, Table 2. Theglycine hinge region between the two RNA binding domains (amino acids95-103) is dissimilar among the three polypeptide sequences. The secondRNA binding domains (104-178) of the three polypeptide sequences have59-65 identical amino acids and 69-70 conserved amino acids out of the75 amino acids, Table 3. The next domain, a serine-arginine rich domain(179-250) has 42/72 S/R residues in zmSRp30, 44/72 S/R residues inzmSRp31, and 45/72 in zmSRp32. In this S/R domain, 35-38 out of 72 aminoacids residues are identical among the three sequences, Table 4. A PSKdomain of approximately 30 amino acids is found at the carboxy terminus.A similar domain is also found in Arabidopsis, but not in mammaliansplicing factors. TABLE 1 Comparison of polypeptide sequences The %identity is the first number in the box. The % conserved is the numberin the parenthesis. zmSRp30 zmSRp31 zmSRp32 zmSRp30 72% (81%) 66% (77%)zmSRp31 72% (81%) 67% (78%) zmSRp32 66% (77%) 67% (78%)

TABLE 2 Comparison of the first RNA binding domain, amino acids 8-94 Thefirst number in the box is the number of identical amino acids. Thesecond number is the number of conserved amino acids. zmSRp30 zmSRp31zmSRp32 zmSRp30 76 (82) 75 (81) zmSRp31 76 (82) 75 (82) zmSRp32 75 (81)75 (82)

TABLE 3 Comparison of the second RNA binding domain, amino acids 104-178The first number in the box is the number of identical amino acids. Thesecond number is the number of conserved amino acids. zmSRp30 zmSRp31zmSRp32 zmSRp30 65 (69) 62 (70) zmSRp31 65 (69) 59 (69) zmSRp32 62 (70)59 (69)

TABLE 4 Comparison of the S/R domain, amino acids 179-250 The firstnumber in the box is the number of identical amino acids. The secondnumber is the number of conserved amino acids. zmSRp30 zmSRp31 zmSRp32zmSRp30 38 (45) 37 (44) zmSRp31 38 (45) 35 (46) zmSRp32 37 (44) 35 (46)

In addition, zmSRp30, zmSRp31 and zmSRp32 are among the first monocotsSRs reported. A putative pre-mRNA rice alternative splicing factor SF2has been annotated in the NCBI protein database as Accession No.BAB90350. An Arabidopsis pre-mRNA splicing factor, SR1, has also beenidentified PNAS 92, (1995) 7672-7675; Lazar.

A comparison of the structural features of two maize SF2/ASF-like genes,zmSR31 and zmSR32 indicates a similar intron/exon architecture. Twelveintrons had been identified for these particular cDNA clones in additionto one intron within the 5′ untranslated region. The first eight exonsencode for a highly conserved two RNA binding domains (RRM). Exons 5 and6 contain the SWQDLKD (SEQ ID NO: 31) motif considered to be a trademarkof all SF2/ASF-like proteins. Exon 4 encodes for a glycine hingeconnecting two RRM domains. This feature is less prominent in zmSRp31and at SRp30. Exonic sequences are split by short introns in the twomaize genes, thus producing an additional exon in maize genes. The threemaize genes contain a long intron separating the exons of the SR domain.The maize SF2/ASF-like genes contain relatively long introns within theRRM domains. The zmSRp3l gene does not have a clear “glycine hinge”feature separating two RRM domains. Also, the phosphorylation domain atthe 3′ end seems not to be present in zmSRp31.

The maize genes are located on two different chromosomes: the zmSRp32was found on chromosome 9, while the zmSRp31 is located on chromosome 6.

The two maize cDNA clones, zmSR32 and zmSR31, show strong′ homology toeach other; 67% identical and 78% similar at the protein level based onthe deduced protein sequence.

Among SR proteins, the subset of the human SF2/ASF-like proteins seemsto play a unique role in the splicing reactions (Kawano, T. et al.,Mech. Dev. (2000) 95:67-76). Knock-outs of the SF2/ASF-like genes inchicken, mouse, Drosophila, or C. elegans do not produce viablephenotypes (Jumaa, H. et al., Curr. Biol. (1999) 9:899-902). The resultssuggest that the expression of these genes is important forembryogenesis, organo- and morphogenesis (Longman, D. et al., EMBO J.(2000) 19:1625-1637). These reports further suggest that if SR splicingproteins are inactivated during embroygenesis, cells are viable but donot undergo differentiation. It is an object of this invention toexpress SR, either transiently, stably or to have SR protein presentduring transformation to modulate embryogenesis. Improving embryoformation may, among other effects, serve to increase cell viability andtherefore increase transformation efficiency. Expression of SR also issuggested to play a role in organo- and morphogenesis (Longman, D. etal., EMBO J. (2000) 19:1625-1637). It is an object of this invention tomodulate the expression of SR to affect organ development.

Plants

The isolated nucleic acids and methods of the present invention can beused over a broad range of plant types, including species from thegenera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago,Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium,Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa,Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum,Sorghum, Picea, and Populus.

Furthermore the isolated nucleic acids and method of the presentinvention can be used in monocots or dicots. They may also be used ingrasses, cereals, cereal grasses (wheat, oat and corn) or oilseeds.

They can also be used in wheat, barley, oats, sorghum, rye, millet,rice, corn, sugar cane, coconut palm, canola, alfalfa, soybean, tobacco,cotton, potato or sunflower. Other plants of this invention includecorn, soybeans, sorghum, sunflower, wheat, rice, alfalfa and canola.Types of corn included are field corn and corn useful for food, feed andfuel, sweet corn and popcorn. Field corn is a plant of this invention.

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids ofRNA, DNA, and analogs and/or chimeras thereof, comprising a splicingfactor polynucleotide.

A. Polynucleotides Encoding A Protein of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, or Conservatively Modified or Polymorphic Variants Thereof

The present invention provides isolated heterologous nucleic acidscomprising a splicing factor polynucleotide, wherein the polynucleotideencodes a splicing factor polypeptide, disclosed herein in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, or conservatively modified or polymorphicvariants thereof. Those of skill in the art will recognize that thedegeneracy of the genetic code allows for a plurality of polynucleotidesto encode for the identical amino acid sequence. Such “silentvariations” can be used, for example, to selectively hybridize anddetect allelic variants of polynucleotides of the present invention.Accordingly, the present invention includes polynucleotides of SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, and silent variations of polynucleotidesencoding a polypeptide of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14.

B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library

As indicated in (b), supra, the present invention provides isolatednucleic acids comprising splicing factor polynucleotides, wherein thepolynucleotides are amplified from a Zea mays nucleic acid library. Zeamays lines B73, PHRE1, A632, BMS-P2#10, W23, and Mo17 are known andpublicly available. Other publicly known and available maize lines canbe obtained from the Maize Genetics Cooperation (Urbana, Ill.).

The nucleic acid library may be a cDNA library, a genomic library, or alibrary generally constructed from nuclear transcripts at any stage ofintron processing. Generally, a cDNA nucleic acid library will beconstructed to comprise a majority of full-length cDNAs. Often, cDNAlibraries will be normalized to increase the representation ofrelatively rare cDNAs.

Total RNA Isolation: Libraries can be made from a variety of maizetissues but for optimal results one should isolate RNA's from activelygrowing cells. Full length cDNA libraries from such rapidly-dividingtissues (or cells at the G1/S boundary) would provide opportunities foridentifying full length, splicing related cDNAs. Full length cDNAlibraries can be constructed using the “Biotinylated CAP Trapper” method(Carninci, P., et al., Genomics 37:327-336, 1996) or the “mRNA CapRetention Procedure” (Edery, I., et al., Molecular and Cellular Biology15:3363-3371, 1995). Full length cDNA libraries can be normalized toprovide a higher probability of sampling genes that express at lowlevels. Examples of cDNA library normalization methods are summarized byBento Soares (Bonaldo, M. F., et al., Genome Research 6:791-806, 1996).

Functional fragments of splicing protein can be identified using avariety of techniques such as restriction analysis, Southern analysis,primer extension analysis, and DNA sequence analysis. Function can alsobe determined by complementing yeast strains known to be mutant forsplicing proteins with maize homologs. Primer extension analysis or Sinuclease protection analysis, for example, can be used to localize theputative start site of transcription of the cloned gene. Ausubel atpages 4.8.1 to 4.8.5; Walmsley et al., “Quantitative and QualitativeAnalysis of Exogenous Gene Expression by the S1 Nuclease ProtectionAssay,” in METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER ANDEXPRESSION.

The general approach of such functional analysis involves subcloning DNAfragments of a genomic clone, cDNA clone or synthesized gene sequenceinto an expression vector, introducing the expression vector into aheterologous host, and relying on an assay system such RNA binding,splicing activity or increased transformation efficiency to identifyclones containing functional fragments and genes. Methods for generatingfragments of a cDNA or genomic clone are well known. In addition,variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like. See, for example, Ausubel,pages 8.0.3-8.5.9. Also, see generally, McPherson (ed.), DIRECTEDMUTAGENESIS: A Practical approach, (IRL Press, 1991). Thus, the presentinvention also encompasses DNA molecules comprising nucleotide sequencesthat have substantial sequence similarity with SEQ ID NO: 1, 3, 5, 7, 9,11, 13 and encode zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32,zmSRp32′, and zmSRp32″.

The present invention also provides subsequences of the nucleic acids.Any number of subsequences can be obtained by reference to SEQ ID NOS:1, 3, 5, 7, 9, 11, or 13 and using primers which selectively amplify,under stringent conditions to: at least two sites to the polynucleotidesof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. A variety of methods for obtaining5′ and/or 3′ ends is well known in the art. See, e.g., RACE (RapidAmplification of Complementary Ends) as described in Frohman, M. A., inPCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H.Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc., SanDiego, 1990), pp. 28-38.); see also, U.S. Pat. No. 5,470,722, andCurrent Protocols in Molecular Biology, Unit 15.6, Ausubel et al., Eds.,Greene Publishing and Wiley-lnterscience, New York (1995). Thus, thepresent invention provides splicing factor polynucleotides having thesequence of the splicing factor gene, nuclear transcript, cDNA, orcomplementary sequences and/or subsequences thereof.

Primer sequences can be obtained by reference to a contiguoussubsequence of a polynucleotide of the present invention. Primers arechosen to selectively hybridize, under PCR amplification conditions, toa polynucleotide of the present invention in an amplification mixturecomprising a genomic and/or cDNA library from the same species.Generally, the primers are complementary to a subsequence of theamplicon they yield. In some embodiments, the primers will beconstructed to anneal at their 5′ terminal end's to the codon encodingthe carboxy or amino terminal amino acid residue (or the complementsthereof of the polynucleotides of the present invention. The primerlength in nucleotides is selected from the group of integers consistingof from at least 15 to 50. Thus, the primers can be at least 15, 18, 20,25, 30, 40, or 50 nucleotides in length. A non-annealing sequence at the5′end of the primer (a “tail”) can be added, for example, to introduce acloning site at the terminal ends of the amplicon.

The amplification primers may optionally be elongated in the 3′direction with additional contiguous nucleotides from the polynucleotidesequences, such as SEQ ID NOS: 1, 3, 5, 7, 9, 11, or 13 from which theyare derived. The number of nucleotides by which the primers can beelongated is selected from the group of integers consisting of from atleast 1 to 25. Thus, for example, the primers can be elongated with anadditional 1, 5, 10, or 15 nucleotides. Those of skill will recognizethat a lengthened primer sequence can be employed to increasespecificity of binding (i.e., annealing) to a target sequence.

The amplification products can be translated using expression systemswell known to those of skill in the art and as discussed, infra. Theresulting translation products can be confirmed as polypeptides of thepresent invention by, for example, assaying for the appropriatecatalytic activity (e.g., specific activity and/or substratespecificity), or verifying the presence of one or more linear epitopesthat are specific to a polypeptide of the present invention. Methods forprotein synthesis from PCR derived templates are known in the art andavailable commercially. See, e.g., Amersham Life Sciences, Inc., Catalog'97, p.354.

C. Polynucleotides Which Selectively Hybridize to a Polynucleotide of(A) or (B)

As indicated, supra, the present invention provides isolated nucleicacids comprising splicing factor polynucleotides, wherein thepolynucleotides selectively hybridize, under selective hybridizationconditions, to a polynucleotide of paragraphs (A) or (B) as discussed,supra. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising thepolynucleotides of (A) or (B). For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated from a Zea maysnucleic acid library. Typically, the cDNA library comprises at least 80%full-length sequences, at least 85% or 90% full-length sequences, and atleast 95% full-length sequences. The cDNA libraries can be normalized toincrease the representation of rare sequences. Low stringencyhybridization conditions are typically, but not exclusively, employedwith sequences having a reduced sequence identity relative tocomplementary sequences. Moderate and high stringency conditions canoptionally be employed for sequences of greater identity. Low stringencyconditions allow selective hybridization of sequences having about 70%sequence identity and can be employed to identify orthologous orparalogous sequences.

D. Polynucleotides Having at Least 60% Sequence Identity with thePolynucleotides of (A), (B) or (C)

As indicated in (d), supra, the present invention provides isolatednucleic acids comprising splicing factor polynucleotides, wherein thepolynucleotides have a specified identity at the nucleotide level to apolynucleotide as disclosed above in paragraphs (A), (B), or (C). Thepercentage of identity to a reference sequence is at least 60% and,rounded upwards to the nearest integer, can be expressed as an integerselected from the group of integers consisting of from 60 to 99. Thus,for example, the percentage of identity to a reference sequence can beat least 70%, 72%, 75%, 76%, 77%, 78%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96% 97%, 98% or 99%.

Optionally, the polynucleotides of this embodiment will share an epitopewith a polypeptide encoded by the polynucleotides of (A), (B), or (C).Thus, these polynucleotides encode a first polypeptide that elicitsproduction of antisera comprising antibodies that are specificallyreactive to a second polypeptide encoded by a polynucleotide of (A),(B), or (C). However, the first polypeptide does not bind to antiseraraised against itself when the antisera has been fully immunosorbed withthe first polypeptide. Hence, the polynucleotides of this embodiment canbe used to generate antibodies for use in, for example, the screening ofexpression libraries for nucleic acids comprising polynucleotides of(A), (B), or (C), or for purification of, or in immunoassays for,polypeptides encoded by the polynucleotides of (A), (B), or (C). Thepolynucleotides of this embodiment embrace nucleic acid sequences thatcan be employed for selective hybridization to a polynucleotide encodinga polypeptide of the present invention.

Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT Patent Publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent Publication Nos. 92/05258,92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

Optionally, the polynucleotides of this embodiment will encode a proteinhaving a specific activity at least 20%, 30%, 40%, or 50% of the native,endogenous (i.e., non-isolated), full-length splicing factorpolypeptide. Further, the proteins encoded by polynucleotides of thisembodiment will optionally have a substantially similar apparentdissociation constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, K_(cat)) as the native endogenous,full-length splicing factor protein. Those of skill in the art willrecognize that k_(cat)/K_(m) value determines the specificity forcompeting substrates and is often referred to as the specificityconstant. Proteins of this embodiment can have a K_(cat)/K_(m) value atleast 10% of the non-isolated full-length splicing factor polypeptide asdetermined using the substrate of that polypeptide from the splicingspecific pathways, supra. Optionally, the k_(cat)/K_(m) value will be atleast 20%, 30%, 40%, 50%, and at least 60%, 70%, 80%, 90%, or 95% thek_(cat)/K_(m) value of the non-isolated, full-length splicing factorpolypeptide. Determination of k_(cat), K_(m), and K_(cat)/K_(m) can bedetermined by any number of means well known to those of skill in theart. For example, the initial rates (i.e., the first 5% or less of thereaction) can be determined using rapid mixing and sampling techniques(e.g., continuous-flow, stopped-flow, or rapid quenching techniques),flash photolysis, or relaxation methods (e.g., temperature jumps) inconjunction with such exemplary methods of measuring asspectrophotometry, spectrofluorimetry, nuclear magnetic resonance, orradioactive procedures. Kinetic values are conveniently obtained using aLineweaver-Burk or Eadie-Hofstee plot.

Optionally the polynucleotides of this invention will encode a proteinhaving RNA binding activity and/or will encode a protein that modulatessplicing activity.

E. Polynucleotides That are Subsequences of the Polynucleotides of(A)-(D)

As supra, the present invention provides isolated nucleic acidscomprising splicing factor polynucleotides, wherein the polynucleotidecomprises at least 15 contiguous bases from the polynucleotides of (A)through (D) as discussed above. The length of the polynucleotide isgiven as an integer selected from the group consisting of from at least15 to the length of the nucleic acid sequence from which thepolynucleotide is a subsequence of. Thus, for example, polynucleotidesof the present invention are inclusive of polynucleotides comprising atleast 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides inlength from the polynucleotides of (A)-(D). Optionally, the number ofsuch subsequences encoded by a polynucleotide of the instant embodimentcan be any integer selected from the group consisting of from 1 to 20,such as 2, 3, 4, or 5. The subsequences can be separated by any integerof nucleotides from 1 to the number of nucleotides in the sequence suchas at least 5, 10, 15, 25, 50, 100, or 200 nucleotides. Optionally, thenumber of nucleotides in a subsequence is a percent of the designatedcoding sequence. For example, polynucleotides of the present inventionare inclusive of polynucleotides comprising at least 50%, 60%, 70%, 80%,90%, 93%, 95%, 97%, 98% or 99% of the coding nucleotides.

The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived. For example, a subsequencefrom a polynucleotide encoding a polypeptide having at least one linearepitope in common with a prototype sequence, may encode an epitope incommon with the prototype sequence. Alternatively, the subsequence maynot encode an epitope in common with the prototype sequence but can beused to isolate the larger sequence by, for example, nucleic acidhybridization with the sequence from which it's derived. Subsequencescan be used to modulate or detect gene expression by introducing intothe subsequence compounds that bind, intercalate, cleave and/orcrosslink to nucleic acids. Exemplary compounds include acridine,psoralen, phenanthroline, naphthoquinone, daunomycin orchloroethylaminoaryl conjugates.

Gene or Trait Stacking

In certain embodiments the nucleic acid sequences of the presentinvention can be combined with polynucleotide sequences of interest inorder to create plants with a desired phenotype. For example, thepolynucleotides of the present invention may be stacked with any otherpolynucleotides or with other useful genes. The combinations generatedcan also include multiple copies of any one of the polynucleotides ofinterest. The polynucleotides of the present invention can also bestacked with any other gene or combination of genes to produce plantswith a variety of desired trait combinations including but not limitedto traits desirable for animal feed such as high oil genes (e.g., U.S.Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat.Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122);and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem.261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989)Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modifiedstorage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7,2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filedDec. 3, 2001)), the disclosures of which are herein incorporated byreference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881; Geiseret al (1986)Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones et al. (1994) Science 266:789; Martin etal. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529 ); modifiedoils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE) and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)),the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides providing agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5,583,210), stalk strength, flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosuresof which are herein incorporated by reference.

These stacked combinations can be created by any method including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. Sequences can have the same 5′ and3′ splice sites or different ones. In certain cases, it may be desirableto introduce a transformation cassette that will suppress the expressionof the polynucleotide of interest. This may be combined with anycombination of other suppression cassettes or over-expression cassettesto generate the desired combination of traits in the plant. A furtherembodiment of this invention is to use the polynucleotides presented inDNA integration recombinase systems. For instance the polynucleotides ofzmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32, zmSRp32′, or zmSRp32″ maybe flanked with recombination sites such as FRT sites and/or Cre sites.See for example U.S. Pat. No. 6,187,994.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot. In some embodiments the monocot is Zea mays includingZea mays tissue from tassel and vegetative meristem.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isgenerally a vector, adapter, or linker for cloning and/or expression ofa polynucleotide of the present invention. Use of cloning vectors,expression vectors, adapters, and linkers is well known in the art.Exemplary nucleic acids include such vectors as: M13, lambda ZAPExpress, lambda ZAP II, lambda gt10, lambda gt11, PBK-CMV, PBK-RSV,pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4, pWE15,SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS±, pSG5, pBK, pCR-Script, pET,PSPUTK, p3'SS, pOPRSVI CAT, pOP13 CAT, pXT1, pSG5, pPbac, pMbac,pMC1neo, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405,pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambdaMOSElox. For a description of various nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996,1997 (La Jolla, Calif.);and, Amersham Life Sciences, Inc., Catalog '97 (Arlington Heights,Ill.).

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. While isolation ofRNA and construction of cDNA and genomic libraries is well known tothose of ordinary skill in the art, the following highlights some of themethods employed.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90-99 (1979); thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979);the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862 (1981); the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers, Tetra. Letts. 22(20)1859-1862(1981), e.g., using an automated synthesizer, e.g., as described inNeedham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984);and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis generally produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polynucleotide of the present invention,for example a cDNA or a genomic sequence encoding a full lengthpolypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

Splicing vectors were constructed using standard molecular biologytechniques. See, for example, Sambrook et al. (eds.) MOLECULAR CLONING:a LABORATORY MANUAL, Second Edition, (Cold Spring Harbor LaboratoryPress, cold Spring Harbor, N.Y. 1989). Plasmids are based on pUC18. Thevectors used in these experiments contain combinations of the same basicregulatory elements. The Omega prime (O′) 5-prime sequence is describedby Gallie et al., Nucl. Acids Res. 15:3257-3273 (1987). The selectivemarker gene, bar (Thompson et al., EMBO J. 6:2519-2523 (1987)), was usedin conjunction with bialaphos selection to recover transformants. TheCauliflower Mosaic Virus 35S promoter with a duplicated enhancer regionis described by Gardner et al., Nucl. Acid Res. 9:2871-2888 (1981). The79 bp Tobacco Mosaic Virus leader is described by Gallie et al., Nucl.Acid Res. 15:3257-3273 (1987) and was inserted downstream of thepromoter followed by the first intron of the maize alcohol dehydrogenasegene ADH1-S. Described by Dennis et al., Nucl. Acid Res. 12:3983-3990(1984). The 3′ sequence pinII is described by An et al., Plant Cell1:115-122 (1989).

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,the GRP1-8 promoter, and other transcription initiation regions fromvarious plant genes known to those of skill.

Promoters

A. Inducible Promoters

An inducible promoter can be operably linked to a nucleotide sequenceencoding zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32, zmSRp32′, orzmSRp32″. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a nucleotide sequence encoding zmSRp30, zmSRp30′, zmSRp31, zmSRp31′,zmSRp32, zmSRp32′, or zmSRp32″. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal. Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude that from the ACE1 system which responds to copper (Mett et al.,PNAS-90:45674571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen.Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genet. 227:229-237 (1991). An inducible promoter is a promoter thatresponds to an inducing agent to which plants do not normally respond.An exemplary inducible promoter is the inducible promoter from a steroidhormone gene the transcriptional activity of which is induced by aglucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci.U.S.A. 88:10421 (1991)) or the ecdysone inducible promoter (U.S. Pat.No. 6,504,082, issued Jan. 7, 2003).

The expression vector comprises an inducible promoter operably linked toa nucleotide sequence encoding zmSRp30, zmSRp30′, zmSRp31, zmSRp31′,zmSRp32, zmSRp32′, or zmSRp32″. The expression vector is introduced intoplant cells and presumptively transformed cells are exposed to aninducer of the inducible promoter. The cells can be screened for thepresence of encode zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32,zmSRp32′, or zmSRp32″protein by northern, RPA, or RT-PCR (usingtransgene specific probes/oligo pairs) BrdU or splicing assays, asdescribed above.

B. Tissue-Preferred Promoters

A tissue-specific promoter can be operably linked to a nucleotidesequence encoding zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32,zmSRp32′, or zmSRp32″protein. Optionally, the tissue-preferred promoteris operably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a nucleotide sequence encoding zmSRp30,zmSRp30′, zmSRp31, zmSRp31′, zmSRp32, zmSRp32′, or zmSRp32″. Plantstransformed with a gene encoding zmSRp30, zmSRp30′, zmSRp31, zmSRp31′,zmSRp32, zmSRp32′, or zmSRp32″operably linked to a tissue-preferredpromoter produce the zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32,zmSRp32′, or zmSRp32″ protein exclusively, or in a specific tissue.

Any tissue-preferred promoter can be utilized in the instant invention.Exemplary tissue-preferred promoters include a seed-preferred promotersuch as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA82:3320-3324 (1985)), napin promoter, β-conglycinin promoter soybeanlectin promoter, maize 15 kD zein promoter, 22 kD zein promoter, γ-zeinpromoter, waxy promoter, shrunken 1 promoter, globulin 1 promoter andshrunken 2 promoter (Thompson et al.; BioEssays; Vol. 10; p. 108;(1989); a leaf and light-induced promoter such as that from cab orrubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko etal., Nature 318:579-582 (1985)); an anther-preferred promoter such asthat from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); apollen-preferred promoter such as that from Zm13 (Guerrero et al., Mol.Gen. Genet. 224:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).

The expression vector comprises a tissue-preferred promoter operablylinked to a nucleotide sequence encoding a splicing factor protein. Theexpression vector is introduced into plant cells. The cells are screenedfor the presence of splicing factor protein by splicing assays, asdescribed above. Tissue-preferred promoters may include maletissue-preferred promoters such as described in U.S. Pat. No. 6,452,069.

C. Constitutive Promoters

A constitutive promoter can be operably linked to a nucleotide sequenceencoding a splicing factor protein or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a nucleotide sequence encoding splicingfactor protein.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include the promoters fromplant viruses such as the 35S promoter from CaMV (Odell et al., Nature313:810-812 (1985)), Commelina yellow mottled virus (R. Torbert et al.,Plant Cell Rep. 17:284-287 (1988)) and the promoters from such genes asrice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genet. 231:276-285 (1992) and Atanassova et al., Plant Journal2(3):291-300 (1992)).

The ALS promoter, a XbaI/NcoI fragment 5-prime to the Brassica napusALS3 structural gene (or a nucleotide sequence that has substantialsequence similarity to the XbaI/NcoI fragment), represents aparticularly useful constitutive promoter. (U.S. Pat. No. 5,659,026.

The expression vector comprises a constitutive promoter operably linkedto a nucleotide sequence encoding splicing factor protein. Theexpression vector is introduced into plant cells and presumptivelytransformed cells are screened for the presence of splicing factorprotein by splicing assays.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack (e.g. PR promoter), anaerobicconditions, or the presence of light. Examples of inducible promotersare the AdhI promoter which is inducible by hypoxia or cold stress, theHsp70 promoter which is inducible by heat stress, and the PPDK promoterwhich is inducible by light.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or in certain tissues, such as leaves,roots, fruit, seeds, or flowers. The operation of a promoter may alsovary depending on its location in the genome. Thus, an induciblepromoter may become fully or partially constitutive in certainlocations.

Both heterologous and non-heterologous (i.e., endogenous) promoters canbe employed to direct expression of the nucleic acids of the presentinvention. These promoters can also be used, for example, in recombinantexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter splicing content and/or composition in adesired tissue. Thus, in some embodiments, the nucleic acid constructwill comprise a promoter functional in a plant cell, such as in Zeamays, operably linked to a polynucleotide of the present invention.Promoters useful in these embodiments include the endogenous promotersdriving expression of a polypeptide of the present invention.

In some embodiments, isolated nucleic acids which serve as promoter orenhancer elements can be introduced in the appropriate position(generally upstream) of a non-heterologous form of a polynucleotide ofthe present invention so as to up or down regulate expression of apolynucleotide of the present invention. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868), or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a splicing factor geneso as to control the expression of the gene. Gene expression can bemodulated under conditions suitable for plant growth so as to altersplicing content and/or composition. Thus, the present inventionprovides compositions, and methods for making, heterologous promotersand/or enhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

Methods for identifying promoters with a particular expression pattern,in terms of, e.g., tissue type, cell type, stage of development, and/orenvironmental conditions, are well known in the art. See, e.g., TheMaize Handbook, Chapters 114-115, Freeling and Walbot, Eds., Springer,New York (1994); Corn and Corn Improvement, 3^(rd) edition, Chapter 6,Sprague and Dudley, Eds., American Society of Agronomy, Madison, Wis.(1988). A typical step in promoter isolation methods is identificationof gene products that are expressed with some degree of specificity inthe target tissue. Amongst the range of methodologies are: differentialhybridization to cDNA libraries; subtractive hybridization; differentialdisplay; differential 2-D gel electrophoresis; DNA probe arrays; andisolation of proteins known to be expressed with some specificity in thetarget tissue. Such methods are well known to those of skill in the art.Commercially available products for identifying promoters are known inthe art such as the Clontech (Palo Alto, Calif.) Universal GenomeWalkerKit.

Once promoter and/or gene sequences are known, a region of suitable sizeis selected from the genomic DNA that is 5′ to the transcriptionalstart, or the translational start site, and such sequences are thenlinked to a coding sequence. If the transcriptional start site is usedas the point of fusion, any of a number of possible 5′ untranslatedregions can be used in between the transcriptional start site and thepartial coding sequence. If the translational start site at the 3′ endof the specific promoter is used, then it is linked directly to themethionine start codon of a coding sequence.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or from any other eukaryotic gene.

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold. Buchman and Berg, Mol. CellBiol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987).Such intron enhancement of gene expression is typically greatest whenplaced near the 5′ end of the transcription unit. Use of maize intronsAdh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. Seegenerally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds.,Springer, N.Y. (1994).

The vector comprising the sequences from a polynucleotide of the presentinvention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptII gene encodesresistance to the antibiotics kanamycin and geneticin, and the ALS geneencodes resistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al.,Meth. In Enzymol. 153:253-277 (1987). These vectors are plantintegrating vectors in that on transformation, the vectors integrate aportion of vector DNA into the genome of the host plant. Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene 61:1-11 (1987) and Berger et al., Proc. Natl. Acad.Sci. USA 86:8402-8406 (1989). Another useful vector herein is plasmidpBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto,Calif.).

A polynucleotide of the present invention can be expressed in eithersense or anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable plant characteristics. Antisensetechnology can be conveniently used to gene expression in plants. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the anti-sense strand of RNAwill be transcribed. The construct is then transformed into plants andthe antisense strand of RNA is produced. In plant cells, it has beenshown that antisense RNA inhibits gene expression by preventing theaccumulation of mRNA which encodes the enzyme of interest, see, e.g.,Sheehy et al., Proc. Nat'l. Acad. Sci. USA 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

Another method of suppression is sense suppression. Introduction ofnucleic acid configured in the sense orientation has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

Proteins

The isolated proteins of the present invention comprise a polypeptidehaving at least 10 amino acids encoded by any one of the polynucleotidesof the present invention as discussed more fully, supra, or polypeptideswhich are conservatively modified variants thereof. Exemplarypolypeptide sequences are provided in SEQ ID NOS: 2, 4, 6, 8, 10, 12,and 14. The proteins of the present invention or variants thereof cancomprise any number of contiguous amino acid residues from a polypeptideof the present invention, wherein that number is selected from the groupof integers consisting of from 10 to the number of residues in afull-length splicing polypeptide. Optionally, this subsequence ofcontiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acidsin length, often at least 50, 60, 70, 80, or 90 amino acids in length.Further, the number of such subsequences can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, or 5.

As those of skill will appreciate, the present invention includes bothbinding and catalytically active polypeptides of the present invention.Catalytically active polypeptides have a specific activity at least 20%,30%, or 40%, and at least 50%, 60%, or 70%, and at least 80%, 90%, or95% that of the native (non-synthetic), endogenous polypeptide. Further,the substrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40%, or 50%, that of thenative (non-synthetic), endogenous polypeptide; and at least 60%, 70%,80%, or 90%. Methods of assaying and quantifying measures of enzymaticactivity and substrate specificity (K_(cat)/K_(m)), are well known tothose of skill in the art.

Generally, the proteins of the present invention will, when presented asan immunogen, elicit production of an antibody specifically reactive toa polypeptide of the present invention encoded by a polynucleotide ofthe present invention as described, supra. Exemplary polypeptidesinclude those which are full-length, such as those disclosed in SEQ IDNOS: 2, 6, and 10. Further, the proteins of the present invention willnot bind to antisera raised against a polypeptide of the presentinvention which has been fully immunosorbed with the same polypeptide.Immunoassays for determining binding are well known to those of skill inthe art. Thus, the proteins of the present invention can be employed asimmunogens for constructing antibodies immunoreactive to a protein ofthe present invention for such exemplary utilities as immunoassays orprotein purification techniques.

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian, or plant cells. The cells producethe protein in a non-natural condition (e.g., in quantity, composition,location, and/or time), because they have been genetically alteredthrough human intervention to do so. In eukaryotic cells overexpressionof a non-functional fusion protein may be desirable. After isolation andpurification of the fusion protein from the expressing cells, enzymaticcleavage could be used to restore function to the purified zmSRp30,zmSRp31, or zmSRp32 protein. In addition, fusions with zmSRp30, zmSRp31or zmSRp32 can have application for affinity matrices and affinitycolumns used for purifying other splicing factor genes. For example,“His-patch” thioredoxin fusions can be expressed, and the isolateHis-zmSRp31 or His-zmSRp32 fusion protein bound to metal chelatecolumns. Whole cell protein extracts can then be passed through thecolumn to selectively trap proteins that interact with zmSRp30, zmSRp31or zmSRp32. See Ausubel et al., 1990 for general methods. Similarly,glutathione-S transferase fusions can be used to attach proteins tosolid-phase matrices for this type of affinity binding. This method hasbeen used, for example, to identify splicing genes whose proteins bindto GST-Rb in L. Magnaghi-Jaulin et al., Retinoblastoma protein repressestranscription by recruiting a histone deacetylase. Nature 391:601-604(1998). It may also be advantageous to fuse additional functional genesto the zmSRp30, zmSRp30′, zmSRp31, zmSRp31′, zmSRp32, zmSRp32′, orzmSRp32″ gene. For example it would be useful to fuse a greenfluorescent gene or some other reporter gene.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible) followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter to direct transcription, aribosome binding site for translational initiation, and atranscription/translation terminator. One of skill would recognize thatmodifications can be made to a protein of the present invention withoutdiminishing its biological activity. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, a methionine addedat the amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

A. Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang et al., Nature 198:1056 (1977)), the tryptophan (trp) promotersystem (Goeddel et al., Nucleic Acids Res. 8:4057 (1980)) and the lambdaderived P L promoter and N-gene ribosome binding site (Shimatake et al.,Nature 292:128 (1981)). The inclusion of selection markers in DNAvectors transfected in E. coli is also useful. Examples of such markersinclude genes specifying resistance to ampicillin, tetracycline, orchloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

B. Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, a of the present invention can beexpressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, F.,et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982)is a well recognized work describing the various methods available toproduce the protein in yeast. Suitable vectors usually have expressioncontrol sequences, such as promoters, including 3-phosphoglyceratekinase or other glycolytic enzymes, and an origin of replication,termination sequences and the like as desired. For instance, suitablevectors are described in the literature (Botstein et al., Gene 8:17-24(1979); Broach et al., Gene 8:121-133 (1979)).

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates. The monitoring of the purification processcan be accomplished by using Western blot techniques or radioimmunoassayof other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cell cultures. Mammalian cell systems often will be in theform of monolayers of cells although mammalian cell suspensions may alsobe used. A number of suitable host cell lines capable of expressingintact proteins have been developed in the art, and include the HEK293,BHK21, and CHO cell lines. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Other animal cells useful for production of proteins of thepresent invention are available, for instance, from the American TypeCulture Collection Catalogue of Cell Lines and Hybridomas (7th edition,1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (See Schneider, J.Embryol. Exp. Morphol. 27:353-365 (1987)).

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. Saveria-Campo, M.,Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector in DNA CloningVol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington,Va., pp. 213-238 (1985).

Transfection/Transformation/Introduction

The method of transformation/transfection is not critical to the instantinvention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod which provides for efficient transformation/transfection orintroduction of DNA, RNA or protein into a cell may be employed.

Gene Transformation Methods

A DNA sequence coding for the desired polynucleotide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength protein or functional fragment will be used to construct arecombinant expression cassette which can be introduced into the desiredplant.

Numerous methods for introducing foreign genes into plants are known andcan be used to insert the splicing gene into a plant host, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., 1993, “Procedure for Introducing Foreign DNA intoPlants,” In: Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, microorganism-mediatedgene transfer such as Agrobacterium (Horsch et al., Science 227:1229-31,1985), electroporation, micro-injection, and biolistic bombardment.

The most widely utilized method for introducing an expression vectorinto plants is based on the use of Agrobacterium. The Ti and Ri plasmidsof A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, 1991, Crit. Rev. Plant Sci. 10:1. Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provide in Gruber et al., supra; Miki et al., supra; andMoloney et al., 1989, Plant Cell Reports 8:238. Methods forAgrobacterium-mediated transformation in rice is disclosed in (Hiei etal., 1994, The Plant Journal 6:271-282) and maize (Ishida et al., 1996,Nature/Biotechnology 14:745-750). Methods for Agrobacterium-mediatedtransformation in sorghum are disclosed in WO 98/49332. Methods forAgrobacterium-mediated transformation in maize are disclosed in WO98/32326. Several methods of plant transformation, collectively referredto as direct gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles (Sanford et al., 1987, Part. Sci. Technol.5:27; Sanford, 1988, Trends Biotech 6:299; Sanford, 1990, Physiol. Plant79:206; Klein et al., 1992, Biotechnology 10:268). Another method forphysical delivery of DNA to plants is sonication of target cells asdescribed in Zang et al., 1991, BioTechnology 9:996. Alternatively,liposome or spheroplast fusions have been used to introduce expressionvectors into plants. See, for example, Deshayes et al., 1985, EMBO J.4:2731; and Christou et al., 1987, PNAS USA 84:3962. Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. See, for example, Hain et al.,1985, Mol. Gen. Genet. 199:161; and Draperetal., 1982, Plant CellPhysiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, for example, Donn et al., 1990, In: Abstracts of theVIIth Int'l Congress on Plant Cell and Tissue Culture (IAPTC), A2-38,page 53; D'Halluin et al., 1992, Plant Cell 4:1495-1505; and Spencer etal., 1994, Plant Mol. Biol. 24:51-61. Microinjection of DNA into wholeplant cells has also been described as has microinjection intoprotoplasts. See, for example in whole cells, Neuhaus et al., 1987,Theor. Appl. Genet. 75:30-36; and in protoplasts, Crossway et al., 1986,Mol. Gen. Genet. 202:179-185; and Reich et al., 1986, Biotechnology4:1001-1004. Another useful basic transformation protocol involves acombination of wounding by particle bombardment, followed by use ofAgrobacterium for DNA delivery, as described by Bidney et al., PlantMol. Biol. 18:301-313 (1992). Useful plasmids for plant transformationinclude PHP9762. The binary backbone for PHP9762 is bin 19. See Bevan,Nucleic Acids Research 12:8711-8721 (1984).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol. 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter 6:165 (1988). Expression of polypeptide codingnucleic acids can be obtained by injection of the DNA into reproductiveorgans of a plant as described by Pena et al., Nature 325:274 (1987).Transformation can also be achieved through electroporation of foreignDNA into sperm cells then microinjecting the transformed sperm cellsinto isolated embryo sacs as described in U.S. Pat. No. 6,300,543 byCass et al. DNA can also be injected directly into the cells of immatureembryos and the rehydration of desiccated embryos as described byNeuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook et al.,in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54(1986).

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, for example, Gruber et al., 1993, “Vectors for PlantTransformation” In: Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton,pages 89-119.

Once a single transformed plant has been obtained by the a recombinantDNA method, e.g., a plant transformed with a desired gene, conventionalplant breeding methods can be used to transfer the structural gene andassociated regulatory sequences via crossing and backcrossing. Ingeneral, such plant breeding techniques are used to transfer a desiredgene into a specific crop plant. In the instant invention, such methodsinclude the further steps of: (1) sexually crossing a transformed plantwith a second non-transformed plant; (2) recovering embryos, seed orother gametogenic material from the cross; and (3) growingtransgene-containing plants from the embryos, seed or gametogenicmaterial.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These include:calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,biolistics, and micro-injection of the DNA directly into the cells. Thetransfected cells are cultured by means well known in the art. Kuchler,R. J., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillilan Publishing Company, New York, pp. 124-176 (1983); andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

Methods for plant regeneration are known in the art and several methodsare provided by Kamo et al., (Bot. Gaz. 146(3):324-334, 1985), West etal., (The Plant Cell 5:1361-1369,1993), and Duncan et al. (Planta165:322-332,1985).

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horschet al., Science 227:1229-1231 (1985). Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys. 38:467486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, New York (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype, (e.g., altered splicing content or composition).

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced nucleic acidsequences.

Transgenic plants expressing the selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

One embodiment is a transgenic plant that is homozygous for the addedheterologous nucleic acid; i.e., a transgenic plant that contains twoadded nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered cell division relative to a control plant (i.e., native,non-transgenic). Back-crossing to a parental plant and out-crossing witha non-transgenic plant are also contemplated.

Synthesis of Proteins

The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in.Peptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicycylohexylcarbodiimide) is known to those of skill.

Purification of Proteins

The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including selective precipitation with such substances as ammoniumsulfate, column chromatography, immunopurification methods, and others.See, for instance, R. Scopes, Protein Purification: Principles andPractice, Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press (1990). For example, antibodies may beraised to the proteins as described herein. Purification from E. colican be achieved following procedures described in U.S. Pat. No.4,511,503. The protein may then be isolated from cells expressing theprotein and further purified by standard protein chemistry techniques asdescribed herein. Detection of the expressed protein is achieved bymethods known in the art and include, for example, radioimmunoassays,Western blotting techniques or immunoprecipitation.

Modulating Splicing Factor Protein Content and/or Composition

The present invention further provides a method for modulating (i.e.,increasing or decreasing) splicing factor protein content or compositionin a plant or part thereof. Modulation can be effected by increasing ordecreasing the splicing factor protein content (i.e., the total amountof splicing factor protein) and/or the splicing factor proteincomposition (the ratio of various splicing monomers in the plant) in aplant. The method comprises transforming a plant cell, transiently orstably, with a recombinant expression cassette comprising apolynucleotide of the present invention as described above to obtain atransformed plant cell. For stably transformed plant cells, growing thetransformed plant cell under plant forming conditions, and inducingexpression of a polynucleotide of the present invention in the plant fora time sufficient to modulate splicing factor protein content and/orcomposition in the plant or plant part.

In some embodiments, plant splicing or selective alternative splicingmay be modulated by altering, in vivo or in vitro, the promoter of anon-isolated splicing factor gene to up- or down-regulate geneexpression. In some embodiments, the coding regions of native splicingfactor genes can be altered via substitution, addition, insertion, ordeletion to decrease activity of the encoded enzyme. See, e.g., Kmiec,U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. And in someembodiments, an isolated nucleic acid (e.g., a vector) comprising apromoter sequence is transformed into a plant cell. Subsequently, aplant cell comprising the promoter operably linked to a polynucleotideof the present invention is selected for by means known to those ofskill in the art such as, but not limited to, Southern blot, geneexpression analysis, DNA sequencing, or PCR analysis using primersspecific to the promoter and to the gene and detecting ampliconsproduced there from. A plant or plant part altered or modified by theforegoing embodiments is grown under plant forming conditions for a timesufficient to modulate splicing protein content and/or composition inthe plant. Plant forming conditions are well known in the art anddiscussed briefly, supra.

In general, content or composition is increased or decreased by at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%,180%, 200%, 250%, 250%, 280%, or 300% relative to a native controlplant, plant part, or cell lacking the aforementioned recombinantexpression cassette. Modulation in the present invention may occurduring and/or subsequent to growth of the plant to the desired stage ofdevelopment. Modulating nucleic acid expression temporally and/or inparticular tissues can be controlled by employing the appropriatepromoter operably linked to a polynucleotide of the present inventionin, for example, sense or antisense orientation as discussed in greaterdetail, supra. Induction of expression of a polynucleotide of thepresent invention can also be controlled by exogenous administration ofan effective amount of inducing compound. Inducible promoters andinducing compounds that activate expression from these promoters arewell known in the art. In other embodiments, splicing is modulated inmonocots, particularly maize.

Molecular Markers

The present invention provides a method of genotyping a plant comprisinga polynucleotide of the present invention. Genotyping provides a meansof distinguishing homologs of a chromosome pair and can be used todifferentiate segregants in a plant population. Molecular marker methodscan be used for phylogenetic studies, characterizing geneticrelationships among crop varieties, identifying crosses or somatichybrids, localizing chromosomal segments affecting monogenic traits, mapbased cloning, and the study of quantitative inheritance. See, e.g.,Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed.,Springer-Verlag, Berlin (1997). For molecular marker methods, seegenerally, The DNA Revolution by Andrew H. Paterson 1996 (Chapter 2) in:Genome Mapping in Plants (ed. Andrew H. Paterson) by Academic Press/R.G. Landis Company, Austin, Tex., pp.7-21.

The particular method of genotyping in the present invention may employany number of molecular marker analytic techniques such as, but notlimited to, restriction fragment length polymorphisms (RFLPs). RFLPs arethe product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. As is well known to those ofskill in the art, RFLPs are typically detected by extraction of genomicDNA and digestion with a restriction enzyme. Generally, the resultingfragments are separated according to size and hybridized with a probe;i.e. single copy probes. Restriction fragments from homologouschromosomes are revealed. Differences in fragment size among allelesrepresent an RFLP. Thus, the present invention further provides a meansto follow segregation of a splicing gene or nucleic acid of the presentinvention as well as chromosomal sequences genetically linked to thesegenes or nucleic acids using such techniques as RFLP analysis. Linkedchromosomal sequences are within 50 centiMorgans (cM), often within 40or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2,or 1 cM of a splicing factor gene.

In the present invention, the nucleic acid probes employed for molecularmarker mapping of plant nuclear genomes selectively hybridize, underselective hybridization conditions, to a gene encoding a polynucleotideof the present invention. In other embodiments, the probes are selectedfrom polynucleotides of the present invention. Typically, these probesare cDNA probes or Pst I genomic clones. The length of the probes isdiscussed in greater detail, supra, but are typically at least 15 basesin length, or at least 20, 25, 30, 35, 40, or 50 bases in length.Generally, however, the probes are less than about 1 kilobase in length.Probes are single copy probes that hybridize to a unique locus in ahaploid chromosome complement. Some exemplary restriction enzymesemployed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein theterm “restriction enzyme” includes reference to a composition thatrecognizes and, alone or in conjunction with another composition,cleaves at a specific nucleotide sequence.

The method of detecting an RFLP comprises the steps of (a) digestinggenomic DNA of a plant with a restriction enzyme; (b) hybridizing anucleic acid probe, under selective hybridization conditions, to asequence of a polynucleotide of the present of the genomic DNA; (c)detecting there from a RFLP. Other methods of differentiatingpolymorphic (allelic) variants of polynucleotides of the presentinvention can be had by utilizing molecular marker techniques well knownto those of skill in the art including such techniques as: 1) singlestranded conformation analysis (SSCP); 2) denaturing gradient gelelectrophoresis (DGGE); 3) RNase protection assays; 4) allele-specificoligonucleotides (ASOs); 5) the use of proteins which recognizenucleotide mismatches, such as the E. coli mutS protein; and 6)allele-specific PCR. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); andchemical mismatch cleavage (CMC). Thus, the present invention furtherprovides a method of genotyping comprising the steps of contacting,under stringent hybridization conditions, a sample suspected ofcomprising a polynucleotide of the present invention with a nucleic acidprobe. Generally, the sample is a plant sample; suspected of comprisinga maize polynucleotide of the present invention (e.g., gene, mRNA). Thenucleic acid probe selectively hybridizes, under stringent conditions,to a subsequence of a polynucleotide of the present invention comprisinga polymorphic marker. Selective hybridization of the nucleic acid probeto the polymorphic marker nucleic acid sequence yields a hybridizationcomplex. Detection of the hybridization complex indicates the presenceof that polymorphic marker in the sample. In some embodiments, thenucleic acid probe comprises a polynucleotide of the present invention.

UTR's and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987))and the 5<G>7 methyl GpppG cap structure (Drummond et al., Nucleic AcidsRes. 13:7375 (1985)). Negative elements include stable intramolecular 5′UTR stem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUGsequences or short open reading frames preceded by an appropriate AUG inthe 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284(1988)). Accordingly, the present invention provides 5′ and/or 3′ UTRregions for modulation of translation of heterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5,10, 20, 50, or 100.

Splice Sites

The recognition of exons and introns in plants is a complex process thatis poorly defined. There are at least three sequence elements withinintrons that determine the intron identity. These include the 5′ splicesite also called the donor site, the 3′ splice site referred to as theacceptor site, and the branch site. The most common plant exon/intronjunction sites are AG/GUAAGU at the 5′ splice site and GCAG/G at the 3′splice site. The branch point with a consensus sequence of CURAY isusually located about 30 bp from the 3'splice site and is separated formthis site by a stretch of U-rich region. A number of splicing factorsinteract with specific elements of the intron sequences to form afunctional complex called a spliceosome that is involved in recognitionand processing of introns. In particular, the present inventiondescribes three new plant splicing factors that recognize and define theintron splicing sites (Reddy, ASN, Critical Reviews in Plant Sciences(2001) 20:523-571). Under natural physiological conditions, the processis highly regulated. Mutations within the splicing sites prevent properprocessing of the intron sequences (Lal, S, Choi J H, Curtis Hannah L(1999) Plant Physiol 120:65-72; Lal et al. (1999) Plant Physiol 121:411418). The present invention provides methods for controlling andregulating the splicing process by using splicing factors that are thesubject of this invention.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingthere from. Sequence shuffling is described in PCT Publication No.W096/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA94:4504-4509 (1997). Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system, and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element, orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be anincreased K_(m) and/or k_(cat) over the wild-type protein as providedherein. In other embodiments, a protein or polynucleotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. The increase in such propertiescan be at least 110%, 120%, 130%, 140% or at least 150% of the wild-typevalue.

Detection of Nucleic Acids

The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of comprising a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of corn. In someembodiments, a splicing gene or portion thereof can be amplified priorto the step of contacting the nucleic acid sample with a polynucleotideof the present invention. The nucleic acid sample is contacted with thepolynucleotide to form a hybridization complex. The polynucleotidehybridizes under stringent conditions to a gene encoding a polypeptideof the present invention. Formation of the hybridization complex is usedto detect a gene encoding a polypeptide of the present invention in thenucleic acid sample. Those of skill will appreciate that an isolatednucleic acid comprising a polynucleotide of the present invention shouldlack cross-hybridizing sequences in common with non-splicing genes thatwould yield a false positive result.

Detection of the hybridization complex can be achieved using any numberof well-known methods. For example, the nucleic acid sample, or aportion thereof, may be assayed by hybridization formats including butnot limited to, solution phase, solid phase, mixed phase, or in situhybridization assays. Briefly, in solution (or liquid) phasehybridizations, both the target nucleic acid and the probe or primer arefree to interact in the reaction mixture. In solid phase hybridizationassays, probes or primers are typically linked to a solid support wherethey are available for hybridization with target nucleic in solution. Inmixed phase, nucleic acid intermediates in solution hybridize to targetnucleic acids in solution as well as to a nucleic acid linked to a solidsupport. In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the various hybridization assay formats: Singeret al., Biotechniques 4(3):230-250 (1986); Haase et al., Methods inVirology, Vol. VII, pp.189-226 (1984); Wilkinson, The theory andpractice of in situ hybridization in: In situ Hybrdization, D. G.Wilkinson, Ed., IRL Press, Oxford University Press, Oxford; and NucleicAcid Hybrdization: A Practical Approach, Hames, B. D. and Higgins, S.J., Eds., IRL Press (1987).

Assays for Compounds that Modulate Enzymatic Activity or Expression

The present invention also provides means for identifying compounds thatbind to (e.g., substrates), and/or increase or decrease (i.e., modulate)the activity of active polypeptides of the present invention. The methodcomprises contacting a polypeptide of the present invention with acompound whose ability to bind to or modulate enzyme activity is to bedetermined. The polypeptide employed will have at least 20%, at least30% or 40%, at least 50% or 60%, and at least 70% or 80% of the specificactivity of the native, full-length splicing polypeptide (e.g., enzyme).Generally, the polypeptide will be present in a range sufficient todetermine the effect of the compound, typically about 1 nM to 10 μM.Likewise, the compound will be present in a concentration of from about1 nM to 10 μM. Those of skill will understand that such factors asenzyme concentration, ligand concentrations (i.e., substrates, products,inhibitors, activators), pH, ionic strength, and temperature will becontrolled so as to obtain useful kinetic data and determine thepresence of absence of a compound that binds or modulates polypeptideactivity. Methods of measuring enzyme kinetics are well known in theart. See, e.g., Segel, Biochemical Calculations, 2^(nd) ed., John Wileyand Sons, New York (1976).

All references cited are herein incorporated by reference.

Although the present invention has been described in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious that certain changes and modifications may be practicedwithin the scope of the appended claims.

Identification and Isolation of Members of the SR Family of Pre-mRNASplicing Factors in Maize

Pioneer's cDNA (EST) database was screened for sequence homologies tothe Arabidopsis thaliana SR1, a member of a serine-arginine-rich proteinfamily of splicing factors. A cluster of 33 ESTs contained the sequencewith homology to the second RNA binding domain, SR domain, and PSKdomain of the SR1 cDNA. An EST was isolated from tassels of the maizeA632 genotype and was subsequently used as a probe for screening agenomic BAC library established from the maize Mo17 genotype. Fouroverlapping BAC clones hybridizing with the probe within a common BamHIDNA fragment were identified. One clone was digested with BamHIaccording to the manufacturer conditions (Life Technologies, GrandIsland, N.Y.) and the restriction fragments were sub-cloned into amodified standard cloning vector. E. coli carrying the BAC fragments wasgrown in Luria-Bertani (LB) medium (Sambrook et al., 1982) and plasmidDNA was extracted. DNA restriction fragments were resolved on agarosegels and transferred onto a nitrocellulose membrane using a Schleicher &Schuell turboblotting system (Schleicher & Schuell, Keene, N. H., USA).AlkPhos Direct labeling and detection kit was used for a probe labelingand hybridization according to the manufacturer's instructions (AmershamPharmacia Biotech, Buckinghamshine, England). Four BamHI BAC restrictionfragments were identified and sequenced. Since the BamHI fragments didnot contain the extended promoter region of the gene, a similarprocedure was repeated for the EcoRI restriction fragments of the cloneDNA. The plasmid DNA preparations were probed with a promoter fragmentgenerated by genomic PCR using the exon 1-specific primers using maizeB73 genomic DNA as a template (Universal Genome Walker Kit, Clontech,Palo Alto, Calif., USA).

Another cluster of 25 cDNAs containing a full-length SR cDNA wasisolated from the elongation zone within a stalk internode in the maizeB73 genotype. Based on the sequence information, two primers weredesigned for genomic amplification of the corresponding coding sequence:CATCCGTCGMGCTGCTCGACCTCGACTCAAG (Seq ID 26) andGCATCAGAGMTMCAATAGCTGCATACTACAA (Seq ID 27). Genomic DNA was isolatedfrom B73 leaves using the cetyltrimethyl-ammonium bromide (CTAB) method(Murray and Thompson, 1980). The Expand High Fidelity PCR system® (RocheApplied Science, Indianapolis, Ind., USA) was used to amplify thegenomic fragment that was subsequently cloned into a vector andsequenced.

Alternative Products of the zmSRp32 Gene in Maize Cells

In Arabidpsis, the long intron 9 of atSRp34 is the site of alternativesplicing that generates five protein isoforms from the same pre-mRNA.The alternative processing of the intron 10 of zmSRp32 was tested inmaize B73 young leaves. A set of nested primers was designed to amplifythe zmSRp32 mRNA message by RT-PCR. The pair of primers specific to thefirst exon and 3′-UTR amplified two visible PCR products indicatingheterogeneity within a pool of the zmSRp32 messages. The same exon 1primer was used together with two intron 10 specific antisense primers,I10a and I10b. While no amplification signal was produced by theexon1/I10b primers, the exon 1/I10a primer set amplified about a 1.3 kbfragment. The RT-PCR product was recovered, cloned, and sequenced toindicate the presence of one additional exon, termed exon 11′. The exon11′ was flanked by the 5′ AGAC and 3′ AGGU splicing sites. The 5′splicing site was preceded by a U/A-rich sequence characteristic ofplant introns. The 11′ exon contains twelve additional codons in framewith the exon 10, which are followed by four stop codons. Prematuretermination of translation may produce truncated versions of zmSRp32.Two ESTs were found in a database containing the 11′ exon sequences aspredicted. One of them was isolated from a 4 day old embryo sac.

ZmSRp32 in pre-mRNA Splicing Reactions in Maize Cells

Maize BMS cells under liquid culture conditions efficiently recognizedand processed the ST-LS1 intron from potato (Vancanneyt, G. et al., Mol.Gen. Genet. (1990) 220:245-250) when integrated into the FLP recombinasecoding sequence (See U.S. Pat. No. 5,929,301). The intron 5′ and 3′splicing sites matched the sequence of the plant consensus-splicingsite: AGGU. The same intron integrated into the gusA coding sequence wasonly partially processed when the 5′ splicing site (a donor site) wasmodified from the AGGU consensus sequence to ACGU. The potato intron isan A/U-rich sequence characteristic of plant introns, however, the 3′end of this intron (around the 3′ splicing site) is markedly missing theU/A motif. Nevertheless, overexpression of zmSRp32 in transientlytransformed BMS cells provided complete splicing of the potato intron inthe gusA gene.

In another experiment, vectors containing RepA with 5′/3′ splicingjunctions of CCGU/AGGA, AGGU/AGGU, CCGU/AGGU or AGGU/AGGA, or a controlwithout an intron, were introduced into maize BMS cells along withexpression cassettes containing zmSRp31, zmSRp32, or a control. Theshort 86 bp intron of the wheat dwarf virus (WDV) replication initiatorprotein (Rep) needs to be spliced in order for the virus to replicate ininfected wheat or maize cells. Table 5 shows the results of theexperiment. The results indicated a 50% splicing efficiency in maize BMScells containing zmSRp31 along with the RepA with the 5′ intron splicingsite, CCGU, and the 3′ splicing site, AGGA.

These results suggest, in this instance, that overexpression of zmSRp31and zmSRp32 in BMS cells produces splicing enhancement when a weaksplice site is present, in this case CCGU/AGGA and CCGU/AGGU. Theexperiment also shows that when the 5′ splice site was not optimal thezmSRp31 and zmSRp32 genes increased splicing efficiency. Because thewheat dwarf virus needs spliced and non-spliced RepA to be fullyinfectious, modulating the splicing activity with zmSRp30, zmSRp31 orzmSRp32 implies a role in disease resistance. TABLE 5 Splicing activityratios-spliced to non-spliced Expression Intron- CCGU/ AGGU/ CCGU/ AGGU/of gene less AGGA AGGU AGGU AGGA Control 1 0.2 0.65 0.25 0.45 (without)zmSRp31 1 0.5 0.67 0.3 0.49 zmSRp32 1 0.4 0.5 0.46 0.48Transformation, DNA Recovery, and GUS Staining

Zea mays Black Mexican Sweet (BMS) suspension cells were transformed byAgrobacterium-mediated transformation procedure as described in Zhao etal., 2002. Briefly, BMS cells (2 ml packed cell volume) and 2.5×10⁸Agrobacterium cells both in ml of N6 medium (4 g/l N6 basal salts, 6.85%sucrose, 1.5 mg/l 2,4-D, 0.69 g/l L-proline, 0.5 mg/l thiamine-HCl, and1× Eriksson's vitamin mix, pH 5.2) were mixed together and stirred on agyratory shaker at 140 rpm for 3 hrs at 27° C. in the dark. Fifty μlsamples of the BMS/Agrobacterium co-cultivation mixtures were placed ondry glass microfiber filters (VWR Scientific Products, 911 CommerceCourt; Buffalo Grove, Ill. 60089), which were then transferred onto theN6 co-cultivation medium similar to the one used for the initialpre-incubations but containing 3% sucrose, 2 mg/l 2,4-D, pH 5.8, andsupplemented with 0.3% agar. Plates were incubated in the dark at 27° C.for 24 hrs. Subsequently, filters were transferred onto the same mediasupplemented with 0.1 g/l carbenicilin. DNA was extracted from BMS cellsharvested 3 days after co-cultivation. DNA was eluted in 0.05 ml ofwater and its concentration was estimated with a dsDNA quantitation kit(Molecular Probes, Eugene, Oreg.). Total DNA (80 ng) was used totransform 40 μl, of library-efficiency DH5{acute over (α)} E. colicompetent cells. Electroporation was done using the Bio-Rad Gene Pulserin 2 mm-wide cuvettes at 2.5 kV with capacitance set for 25 μF andresistance of 200 ohmes. Electroporated cells were incubated in 600 μlof 2×YT media at 37° C. for 30 min. After incubation, 200 μl, sampleswere dispensed onto plates containing LB medium supplemented with 0.1g/l ampicilin. The plates were incubated overnight at 37° C. The numberof recovered colonies per plate were averaged for each treatment. ForGUS staining, three days old BMS cells still attached to the filterswere transferred into Petri dishes containing 0.5 ml of X-Gluc solution(1.36 g NaH₂PO₄, 1.74 g Na₂HPO₄, 164 mg K₄Fe(CN)₆.3H₂O, 211 mgK₃Fe(CN)₆, 0.06 ml Triton X-100, 50 mg X-Gluc, pH 7.0, final volume 100ml) (Jefferson et al., 1987). After sealing with parafilm, the plateswere incubated overnight at 37° C. in the dark.

RT-PCR

Three days after transformation BMS cells were collected, frozen inliquid nitrogen, and stored at −80° C. until needed. The cells (100 mg)were homogenized in liquid nitrogen using mortar and pestle, and thenRNA was extracted with the TRIzol reagent (Cat. No.15596018) accordingto the manufacturer's instructions (Invitrogen Life Technologies,Carlsbad, Calif., USA). Usually about 5 to 7 ug RNA was extracted from100 mg cells. RNA was dissolved in water by incubating for 10 min at 55°C. and 5 ul of DNasel (Cat. No. 18068015, Invitrogen Life Technologies,Carlsbad, Calif., USA) was added to eliminate any DNA contamination. Theincubation was for 2 hrs at room temperature. Fifty ng of RNA was usedas a template to amplify the gusA intron region with the exon 1-specificprimer GTCACTCATTACGGCAAAGTGTGGGTCMT (Seq ID 15) and the exon 2-specificprimer GCTTTTTCTTGCCGTTTTCGTCGGTA (Seq ID 16). The Titan RT-PCR kit(Roche Applied Science, Indianapolis, Ind., USA) was used to carry outthe RT-PCR reactions under the following conditions: 50° C. for 30 min,94° C. for 2 min, 10 cycles at 94° C. for 10 sec, 60° C for 30 sec, 68°C. for 1 min, and 25 cycles at 94° C. for 10 sec, 60° C. for 30 sec, 68°C. for 1 min (cycle elongation of 5 sec for each cycle). The products ofthe RT-PCR reaction were separated on 1% agarose gels. The same RNAextraction and RT-PCR protocol was used to isolate and amplify RNA formmaize B73 leaf tissue. The zmSRp32 exon1-specific primerATGAGCAGGCGCTGGAGCCGCACGATCTA (Seq ID 17) was used in combination withthe following primers: GCCACCAAAGCCACTTGMCGATCATG (exon 4) (Seq ID 18),GMGMGGCAGTCCAGTGACAAG (exon 5) (Seq ID 19), GAGAGAAATTATTGATGGATTITTCTG(intron 6b) (Seq ID 20), GAAAACCAAGCMCCGAATGAAATAAAC (intron 6a) (Seq ID21), GACCTTGMCGAGMGAAACAGATCTTG (exon 10) (Seq ID 22),GCTTTlMCAACTTGGTTCCAAAAGCCATGATG (intron 10a) (Seq ID 23),GACATTAGGTAAAATAATGGGACGATTTTAG (intron 10 b) (Seq ID 24), andGTTMGAAAAGMGAGCTCCTGACTCCATC (3'UTR close to the stop codon) (Seq ID25). The product of amplification with the primer pair exon/intron 10awas sequenced.

Vector Construction

Although other vectors may be utilized, many of the Agro vectorsdescribed herewithin contain the pSB11 plasmid backbone integrated intothe super-binary vector pSB1 residing in Agrobacterium strain LBA 4404(Komari, T. et al., Methods of genetic transformation: Agrobacteriumtumefaciens, In: Vasil IK (ed) Molecular Improvement of Cereal Crops,Kluwer Academic Publishers (1999) pp. 43-82). A zmSRp32 expressionvector was constructed by insertion of the 1.3 kb SmaI/SnaBI restrictionfragment containing zmSRp32 under control of the maize ubiquitin-1promoter (Christensen, A. H. et al., Transgenic Res. (1996) 5:213-218)and the potato protease II terminator (An et al., 1989). A GUSexpression vector contained GUS coding sequence with the ST-LS1 intronfrom potato (Vancanneyt, G. et al., Mol. Gen. Genet. (1990) 220:245-250)ligated in the same orientation and position as for the construction ofzmSRp32. he pWI-11 vector was the source of the wheat dwarf virus (WDV)initiator protein gene (rep) (Ugaki, M. et al., Nucl. Acids Res. (1991)19:371-377). A LIR:Rep-containing NheI/SphI fragment was subcloned intothe corresponding restriction sites of a cloning vector and subsequentlymoved into the pSB11 plasmid backbone as an XbaI/PacI restrictionfragment which was then open with BstEII in order to insert the BstEIIfragment containing the zmSRp32 expression unit. This was thenintegrated into the Agrobacterium pSB1 plasmid.

EXAMPLE 1 Experiment to Show that zmSR Expression EnhancesTransformation Efficiency

To demonstrate that zmSRp expression enhances transformation efficiency,a zmSR gene is cloned into a cassette with a constitutive promoter (i.e.either a strong maize promoter such as the ubiquitin (UBI) promoterincluding the first ubiquitin intron, or a weak constitutive promotersuch as nos). Delivery of the zmSR gene in an appropriate plantexpression cassette (for example, in a UBI:: zmSRp31 orzmSRp32::pinII-containing plasmid) cotransformed with 35S::bar::pinIIcan be accomplished through numerous well-established methods for plantcells, including for example particle bombardment, sonication, PEGtreatment or electroporation of protoplasts, electroporation of intacttissue, silica-fiber methods, microinjection or Agrobacterium-mediatedtransformation. Using one of the above methods, DNA is introduced intomaize cells capable of growth on suitable maize culture medium. Suchcompetent cells can be from maize suspension culture, callus culture onsolid medium, freshly isolated immature embryos or meristem cells.Immature embryos of the Hi-II genotype may be used as the target forco-delivery of these two plasmids.

To assess the effect on transgene integration, growth ofbialaphos-resistant colonies on selective medium is a reliable assay.Within 1-7 days after DNA introduction, the embryos are moved ontoculture medium containing 3 mg/l of the selective agent bialaphos.Embryos, and later callus, are transferred to fresh selection platesevery 2 weeks. After 6-8 weeks, transformed calli are recovered.Transgenic callus containing the introduced genes can be verified usingPCR and Southern analysis. Northern analysis can also be used to verifywhich calli are expressing the bar gene, and whether the zmSR gene isbeing expressed at levels above normal wild-type cells (based onhybridization of probes to freshly isolated mRNA population from thecells). In immature embryos that have transient, elevated zmSRexpression, higher numbers of stable transformants are recovered (likelya direct result of increased integration frequencies). Increasedtransgene integration frequency can also be assessed using suchwell-established labeling methods such as in situ hybridizationproviding an improved cellular/molecular environment for this event tooccur.

EXAMPLE 2 Experiment to Show Over-Expression of a Maize AlternativeSplicing Factor in Wheat Increases Resistance to Pre-Harvest Sprouting

Vivipary is a physiological condition that leads to germination ofembryos while attached to the cob. The viviparous-1 (Vp-1) gene appearsnecessary for the maturation phase of seed development. The gene encodesa transcription factor which pre-mRNA transcripts are frequentlymisspliced reducing the capacity to produce a full-length, functionalproteins (McKibbin, R. S. et al., Proc. Natl. Acad. Sci. USA (2002)99:10203-10208). This is particularly severe in hexaploid bread wheatand has a profound effect on the bread-making quality of wheat.Providing additional alternative splicing factor activity during embryodevelopment and maturation may inhibit or prevent vivipary.

The maize alternative splicing factor gene, ZmSR, is cloned into aconstitutive expression cassette (for example, UBI:ubiintron:ZmSRp32::pinII). Wheat immature embryos are transformed usingparticle bombardment with a 35S::bar::35S-containing plasmid; with orwithout co-bombardment of the ZmSR-containing plasmid. Embryogeniccultures are selected on 4 mg/l gluphosinate ammonium and plantsregenerated following published methods (Rosco-Gaunt et al., 2001,incorporated by reference). Plants are grown to maturity and allowed toset seed. Ripe ears are harvested from plants from each treatment (ZmSRand control) at 14 weeks post-anthesis and are placed on moist papertowels on a free-draining surface in a mist propagator. For each ear,the number of nonsprouted and sprouted grains are scored after 7 days.

Another example of decreasing pre-harvest sprouting is to constructalternative splicing factor expression vectors with the zmSR expressionvector designed for expression in the developing embryo. This vectorcontains the GLB1 (globin) promoter driving expression of a zmSR geneand a PinII terminator. In addition, the CAMV35S enhancer and promoterdrive GAT expression and are excisable via FRT sites. GAT genes encodeglyphosate N-acetyltransferase (GAT). See PCT publication WO02/36782 andU.S. application Ser. No.10/427,692. The maize globulin-1 promoter isattached to the coding sequences of zmSR. This promoter assures strongand localized expression of the splicing factors in the developingembryos. The expression vectors are introduced into plant cells bytransformation methods described in the previous examples. Transgenicplants are regenerated from selected calli (for example, byincorporating the bar gene into the expression vectors and growingcallus on media containing 3 mg/l Bialaphos). Selected calli can beanalyzed for the presence of new DNA sequences using commonly knownmethods. After self-pollination of T0 transgenic plants, the T1 embryoscan be analyzed for the presence of alternatively spliced products ofthe Vp-1 gene by RT-PCR method. Western analysis using a polyclonalantibodies can be carried out to identify different isoforms of the Vp-1protein. Embryos from transgenic plants showing the highest % of fullyspliced Vp-1 transcripts are tested for homozygosity by the quantitativePCR method and germinated to produce T1 plants. The grain sprouting testcan be performed on T2 seed generation by placing them on paper towelson a free-draining flat surface in an incubator with humidifier. Thenumbers of nonsprouted and sprouted grains can be scored after about 7days.

EXAMPLE 3 Experiment to Show Over-Expression of a Maize AlternativeSplicing Factor in Wheat Increases Embryogenic Callus Frequency

Wheat immature embryos are transformed using the method of Example 2with an inducible promoter operably linked to a zmSR-containing codingsequence. A comparison between the mean callus scores (percentage of thesurface area with embryogenic callus) of the control and of theZmSR-bombarded scutellar tissues of wheat is predicted to show asignificant improvement in the ZmSR-bombarded scutellar tissues over thecontrol tissues. Furthermore, in examining the quality of embryogeniccalli formed, the ZmSR-bombarded lines will show significant increasesin the number of ‘good’ calli produced. The calli containing azmSR-containing coding sequence would be expected to be generallylarger, more rapidly growing and vigorous (i.e. calli with scores of 3or 4).

The shoot regenerability of cultures can be correlated with the qualityand quantity of somatic embryos produced in each callus. Shootregenerability of zmSR-bombarded calli can be compared to the shootregenerability of the control.

EXAMPLE 4 zmSR Genes in Positive Selection of Wheat Transformants

Transformation a cassette containing a zmSR gene is described in Example2. “Good’ rated calli are visually-selected on the basis of vigorousembryogenic growth and analyzed by PCR for the presence of theUbi::zmSR::pinII transcription unit. Thus, transformed lines may beidentified without selection. Typically, without chemical selection suchtransformed embryogenic calli cannot be recovered, except perhaps viapositive selection for example with GFP.

When the zmSR gene is introduced, or co-introduced, into a plant cellwithout any additional selective marker, transgenic calli are identifiedby their ability to grow more rapidly than surrounding wild-type(non-transformed) tissues. Transgenic callus can be verified using PCRand Southern analysis. Northern analysis can also be used to verifywhich calli are expressing the maize SR gene at levels above normalwild-type cells (based on hybridization of probes to freshly isolatedmRNA population from the cells).

The zmSR genes can also be cloned into a cassette with an induciblepromoter such as the benzenesulfonamide-inducible promoter. Theexpression vector is co-introduced into plant cells and after selection,as described above or on bialaphos, the transformed cells are exposed tothe safener (inducer). This chemical induction of zmSR expression shouldresult in increased embryogenesis and increased transformationefficiency. The cells are screened for the presence of the zmSR RNA bynorthern, or RT-PCR (using transgene specific probes/oligo pairs eg),for zmSR-encoded protein using zmSR-specific antibodies in Westerns orusing hybridization. Likewise, other assays could be employed.

EXAMPLE 5 Control of Gene Expression Using Tissue-Preferred orCell-Preferred Promoters

ZmSR gene expression using tissue-preferred or cell-preferred promotersmodulates splicing in the corresponding tissues or cells. For example,using a seed-preferred promoter will stimulate cell division rate andresult in increased seed biomass. Alternatively, driving zmSR expressionwith a strongly-expressed, early, tassel-preferred promoter will enhancedevelopment of this entire reproductive structure.

Expression of zmSR genes in other cell types and/or at different stagesof development will similarly stimulate cell division rates. Similar toresults observed in Arabidopsis (Doerner et al., 1996), root-preferredor root-preferred expression of zmSR is predicted to result in largerroots and faster growth (i.e. more biomass accumulation).

EXAMPLE 6 Modulating Flowering Time

Alternative splicing is involved in the control of flowering time. TheArabidopsis floral promoter FCA is alternatively spliced to produce atleast four different transcripts. The accurate excision of all 21introns is required to generate the gamma (γ) transcript, the onlyfull-length transcript producing functional protein. There is less than1% of transcript alpha (α), 55% transcript beta (β), 35% transcriptgamma (γ), and transcript delta (δ) accounts for about 10% of the FCAmRNA in seedlings. Overexpression of the gamma (γ) transcript (providedas an FCA-gamma (γ) cDNA clone controlled by the 35S promoter) causesthe plant to flower significantly earlier than the wild type (Macknight,R. et al., Plant Cell (2002) 14:877-888).

Overexpression of the alternative splicing factors zmSRp30, zmSRp31 andzmSRp32 of this invention can alter the relative abundance of thetranscripts resulting in modulation of the flowering time. An expressionvector containing zmSR under control of a strong plant promoter (such asthe maize ubiquitin promoter) is introduced into plant cells byAgrobacterium-mediated transformation of immature embryos. Since it hasbeen established that the alternative splicing factors act in aconcentration-dependent manner, overexpression of such factors willincrease processing of the pre-mRNAs synthesized from the endogenous FCAgene, in particular, it will lead to accumulation of a fully-splicedtranscripts encoding the functional FCA protein.

To detect if alternative splicing of the FCA messages takes place in thetransgenic cells, the reverse transcriptase-mediated polymerase chainreaction (RT-PCR) can be used to identify different FCA transcripts.These observations will be correlated with the evaluation of theflowering time under different environmental conditions (for example,plus/minus vernalization, long/short-day light exposures). Additionalsplicing of the FCA messages catalyzed by overexpressed alternativesplicing factors will lead to earlier flowering of plants carrying thetransgene.

Alternatively, an inducible promoter such as thebenzenesulfonamide-inducible promoter can control the expression of thealternative splicing factor genes. The expression vector isco-introduced into plant cells and after selection transgenic plants areregenerated. At the time of desired flowering, the transgenic plants areexposed to the safener (inducer). This chemical induction of thealternative splicing factors expression should result in morefully-spliced floral promoter messages and earlier flowering.

EXAMPLE 7 A Method of Affecting Starch Composition

A type I starch-branching enzyme (SBEI) is able to produce three formsof SBEI gene transcripts by alternative splicing of the primary mRNA.This enzyme catalyzes the formation of amylopectin by introducing branchpoints into the linear amylose molecules. Thus, SBEs are important forthe quality of starch synthesized in plants. Selective inhibition ofdifferent isoforms of starch branching enzyme has a profound effect onstarch composition (Baga, M. et al., Plant Mol. Biol. (1999)40:1019-1030; (Itoh, K. et al., Mol. Gen. Genet. (1997) 255:351-358). Inrice, the Waxy gene encodes a granule-bound starch synthase (GBSS). Allrice cultivars described as a low-amylose rice varieties use alternatesplice sites in the GBSS pre-mRNA. They all have mutated sequence(AGTTATA) at the putative leader intron 5′ splice site of the GBSS gene(Frances, H. et al., Plant Mol. Biol. (1998) 38:407-415). Thissingle-base mutation reduces the efficiency of CBSS pre-mRNA processing.

Overexpression of the alternative splicing factors described in thisinvention can alter the relative abundance of the SBEI and the GBSStranscripts resulting in the modification of the starch content. Theexpression unit containing the alternative splicing factors undercontrol of an endosperm promoter, such as the maize gamma (γ) zein GZpromoter, can be introduced into plant cells by methods known to one ofskill in the art.

The ZmSR expression vector designed for expression in the developingendosperm has Gamma Zein (GZ) promoter driving the zmSR gene and aGZUBI1ZM terminator. Also in the vector is the Ubi promoter (Christensenet al., Plant Mol. Biol. 12:619-632 (1.989) and Christensen et al.,Plant Mol. Biol. 18:675-689 (1992)) followed by GAT with a PinIIterminator and flanked by FRT sites (2 on left and 1 on right).

Northern blot analysis of RNA isolated from transgenic events can beused to verify expression of the alternative splicing factors in theendosperm as well as provide evidence for an altered expression of theSBEI messages. The amylose content can be determined by potentiometriciodine titration and the starch content by gel permeationchromatography. Methods of detecting and analyzing relative abundance oftranscripts are known to one of skill in the art and also includeNorthern, PCR and S1 protection.

EXAMPLE 8 Increasing Abiotic Stress Tolerance

Abiotic stress conditions such as drought and salinity strongly affectcrop yields around the world. Pre-mRNA processing seems to be a generaltarget of salt toxicity in eukaryotes (Forment, J. et al., Plant J.(2002) 30:511-519). In particular, members of a family of SR proteinsare known to confer increased tolerance to salts in Arabidopsis. Twoalternative splicing factors described in this patent application aremembers of the plant SR protein family. Transgenic plants, containingthe alternative splicing factor genes, or just part of the codingsequences carrying the SR domain (complete or partial), can be obtainedby genetic transformation using methods described elsewhere. Strongplant promoters, such as the cauliflower mosaic virus 35S promoter orthe maize ubiquitin-1 promoter provide a relatively high level ofprotein expression from the introduced constructs. Primarytransformation events (T0 plants) are regenerated from selectedtransgenic material and selfed to produce homozygous T1 plants.Transformation events are selected based on Southern blot analysis andquantitative PCR-based homozygosity tests. The level of proteinexpression (proteins containing the SR domain) is assessed by Northernblot analysis. Also, a transgene segregation ratio in the T1 generationcan provide information on the transgene integration pattern.

Seeds/kernels from selected transgenic plants and untransformed controlsare germinated in pots for about two-three weeks. After this initialperiod, the seedlings are watered with the 200 mM NaCl solution for thesalt tolerance test. The salt treatment is continued until thesalt-stress symptoms become evident on the control (untransformed)plants. A direct comparison between transgenic plants and salt-stressedcontrol seedlings should identify the salt-tolerant phenotypes. Selectedplants are allowed to grow and set seeds for similar tests in the T2generation.

EXAMPLE 9 Excision of the zmSR Cassette

In cases where the zmSR gene has been stably integrated into a plant andexpression is useful in the recovery of trangenics, but is ultimatelynot desired in the final product, the zmSR expression cassette (or anyportion thereof that is flanked by appropriate FRT recombinationsequences) can be excised using FLP-mediated recombination (see U.S.Pat. No. 5,929,301) or any other excision methods known to one of skillin the art. (See, for example, U.S. Pat. No. 6,187,994; Schlake and Bode(1994) Biochemistry 33:12746-12751; Huang et al. (1991) Nucleic AcidsResearch 19:443448; Paul D. Sadowski (1995) In Progress in Nucleic AcidResearch and Molecular Biology vol. 51, pp. 53-91; Michael M. Cox (1989)In Mobile DNA, Berg and Howe (eds) American Society of Microbiology,Washington D.C., pp. 116-670; Dixon et al. (1995) 18:449-458; Umlauf andCox (1988) The EMBO Journal 7:1845-1852; Buchholz et al. (1996) NucleicAcids Research 24:3118-3119; Kilby et al. (1993) Trends Genet.9:413-421: Rossant and Geagy (1995) Nat Med. 1: 592-594; Albert et al.(1995) The Plant J. 7:649-659: Bayley et al. (1992) Plant Mol. Biol.18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378; and Daleand Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; all of whichare herein incorporated by reference); Lox (Albert et al. (1995) PlantJ. 7:649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA 91:1706-1710;Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Odell et al. (1990)Mol. Gen. Genet. 223:369-378; Dale et al. (1990) Gene 91:79-85; andBayley et al. (1992) Plant Mol. Biol. 18:353-361.)

EXAMPLE 10 The Effect of SR on Treatment of Co-Delivered Transgenes

The plasmids listed in Table 6 below are used to evaluate the influenceof SR on transient expression of co-delivered transgenes; the SuperMASpromoter is that described by Ni et al., 1996; sequence-specificinteractions of wound-inducible nuclear factors with mannopine synthase2′ promoter wound responsive elements, Plant Mol. Biol. 30:77-96. Thevisible marker genes, GUS (b-glucoronidase; Jefferson R. A., Plant Mol.Biol. Rep. 5:387,1987) and GFP (green fluorescent protein; Chalfie etal., Science 263:802,1994) have been described, as has themaize-optimized GFP (GFPm; see U.S. Pat. No. 6,486,382). The Ubiquitinpromoter has been described (Christensen et al., Plant Mol. Biol.12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689(1992), as have the pinII (An et al., 1989, Plant Cell 1:115-122) and35S (Odell et al., 1985, Nature 313:810-812) 3′ regions in theseexpression cassettes. TABLE 6 Constructs used to evaluate the effect ofSR expression on transient expression of co-delivered transgenes PlasmidDescription SuperMAS::GUS::pinll 3′ region UBI::moPAT::CaMV35S 3′ regionUBI::GFPm::pinll WDV-LIR promoter::SRGFP Expression in Maize

Transformation of the zmSR plasmid DNA into an inbred follows awell-established bombardment transformation protocol used forintroducing DNA into the scutellum of immature maize embryos (Songstad,D. D. et al., In Vitro Cell Dev. Biol. Plant 32:179-183,1996). It isnoted that the any suitable method of transformation can be used, suchas Agrobacterium-mediated transformation and many other methods. Cellsare transformed by culturing maize immature embryos (approximately1-1.5mm in length) onto medium containing N6 salts, Erikkson's vitamins,0.69 g/l proline, 2 mg/l 2,4-D and 3% sucrose. After 4-5 days ofincubation in the dark at 28° C., embryos are removed from the firstmedium and cultured onto similar medium containing 12% sucrose. Embryosare allowed to acclimate to this medium for 3 h prior to transformation.The scutellar surface of the immature embryos is targeted using particlebombardment with either a UBI::GFPm::pinII plasmid+aUBI::maize-optimized PAT::pinII plasmid (control treatment) or with acombination of the UBI::GFPm::pinII plasmid+the zmSR plasmid. Embryosare transformed using the PDS-1000 Helium Gun from Bio-Rad at one shotper sample using 650PSI rupture disks. DNA delivered per shot averagedat 0.0667 ug. An equal number of embryos per ear are bombarded witheither the control DNA mixture or the zmSR/GFP DNA mixture. Followingbombardment, all embryos are maintained on standard maize culture medium(N6 salts, Erikkson's vitamins, 0.69 g/l proline, 2 mg/l 2,4-D, 3%sucrose) for 2-3 days and then evaluated for transient GFP expression.

In both experiments, we expect a greater number of cells transientlyexpressing GFP on the scutellar surface in the treatment containing thezmSR DNA when compared to the control.

Soybean

Tissue is excised from coyledons and placed on MS-based medium. Amixture of plasmid DNA, containing equal amounts of aSuperMas::GUS::pinII plasmid and the WDV-LIR::SR plasmid, is deliveredinto cells on the surface of the colyledon explants usingparticle-mediated delivery similar to that described for maize above. Asa control, SuperMas::GUS::pinII plasmid+UBI::moPAT::CaMV35S isintroduced into the same target cells using an equal number ofcotyledonary tissue pieces.

In the SR-treatment, greater numbers of transiently expressing cellswould be expected on the cotyledon after GUS staining. In addition, forcells exhibiting transient gene expression, the level of expression asjudged by relative intensity of histochemical staining would be expectedto be greater in SR-treated tissues (as compared to controls).

EXAMPLE 11 zmSRp31 Increases Growth Rates in Early-Developing StableMaize Transformants

Transformation of the zmSR plasmid DNA into Hi-II follows the standardHi-II bombardment transformation protocol (Songstad D. D. et al., InVitro Cell Dev. Biol. Plant 32:179-183,1996). Cells are transformed byculturing maize immature embryos (approximately 1-1.5 mm in length) onto560P medium containing N6 salts, Erikkson's vitamins, 0,69 g/l proline,2 mg/l 2,4-D and 3% sucrose. After 4-5 days of incubation in the dark at28° C., embryos are removed from 560P medium and cultured, scutellum up,onto 560Y medium which is equivalent to 560P but contains 12% sucrose.Embryos are allowed to acclimate to this medium for 3 h prior totransformation. The scutellar surface of the immature embryos istargeted using particle bombardment with either a UBI::moPAT˜GFPm::pinIIplasmid or with a combination of the UBI::moPAT˜GFPm::pinII plasmid+theGZ::zmSR::35S:GZ′ plasmid. Embryos are transformed using the PDS-1000Helium Gun from Bio-Rad at one shot per sample using 650PSI rupturedisks. DNA delivered per shot averaged at 0.0667 ug. An equal number ofembryos per ear are bombarded with either the control DNA (PAT˜GFP) orthe zmSRp31/PAT˜GFP DNA mixture. Following bombardment, all embryos aremaintained on 560L medium (N6 salts, Eriksson's vitamins, 0.5 mg/lthiamine, 20 g/l sucrose, 1 mg/l 2,4-D, 2.88 g/l proline, 2.0 g/lgelrite, and 8.5 mg/l silver nitrate). After 2-7 days post-bombardment,all the embryos from both treatments are transferred onto N6-basedmedium containing 3 mg/l bialaphos Pioneer 560P medium described supra,with no proline and with 3 mg/l bialaphos). Plates are maintained at 28°C. in the dark and are observed for colony recovery with transfers tofresh medium occurring every two weeks. Two weeks after DNA delivery,the newly-forming callus is examined using epifluorescence under thedissecting microscope (using commercially-available filter combinationsfor GFP excitation and emission).

At 2 weeks post-bombardment, numerous cells on the surface of thescutellar-derived tissue are expected to express GFP in the controltreatment (no zmSRp3l), but expressing foci consisted of single cell. Nomulticellular GFP-expressing clusters are expected in the control. Atthis same time-point, 2-weeks after DNA-delivery, the same sprinkling ofsingle-celled GFP-expressing foci are expected on the surface of thetissue that had received the zmSR/PAT˜GFP mixture. However, numerousmacroscopic GFP-expressing multicellular clusters are also apparent.Many embryos are observed with multiple transgenic microcalli developingon the surface, with independent transformants beginning to grow from asingle embryo.

After 3 weeks, GFP-expressing single cells will be observed in bothtreatments, although the frequency will decline. In the zmSR-treatedembryos, the growth rate of the developing transgenic calli willcontinue to be very rapid. At 5 weeks post-bombardment, many zmSRcolonies will continue to grow rapidly.

EXAMPLE 12 Experiment to Show How Transient zmSR Activity EnhancesTransformation Frequency

For transient zmSR-mediated cell cycle stimulation to increase transientintegration frequencies, it may be desirable to reduce the likelihood ofectopic stable expression of the zmSR gene. Strategies fortransient-only expression can be used. This includes delivery of RNA(transcribed from the zmSR gene), chemically end-modified DNA expressioncassettes that typically will not integrate, or zmSR protein along withthe transgene cassettes to be integrated to enhance transgeneintegration by transient stimulation of cell division. Usingwell-established methods to produce zmSR-RNA, this can then be purifiedand introduced into maize cells using physical methods such asmicroinjection, bombardment, electroporation or silica fiber methods.For protein delivery, the gene is first expressed in a bacterial orbaculoviral system, the protein purified and then introduced into maizecells using physical methods such as microinjection, bombardment,electroporation or silica fiber methods.

Alternatively, zmSR proteins are delivered from Agrobactedum tumefaciensinto plant cells in the form of fusions to Agrobacterium virulenceproteins. Fusions are constructed between zmSR and bacterial virulenceproteins such as VirE2, VirD2, or VirF which are known to be delivereddirectly into plant cells. Fusions are constructed to retain both thoseproperties of bacterial virulence proteins required to mediate deliveryinto plant cells and the zmSR activity required for enhancing transgeneintegration. This method ensures a high frequency of simultaneousco-delivery of T-DNA and functional zmSR protein into the same hostcell. The methods above represent various means of using the zmSR geneor its encoded product to transiently modulate gene expression throughsplicing, which in turn enhances transformation efficiency by providingan improved cellular/molecular environment for this event to occur(WO99/61619).

EXAMPLE 13 Experiment to Show How Re-Transformation of SR-TransgenicProgeny can Result in Elevated Transformation Frequency

Agrobacterium mediated transformation. As the starting point forAgrobacterium-mediated re-transformation experiments, regenerated inbredmaize T0 transformants are produced containing maize SR expressioncassettes and UBI::moPAT˜GFP::pinII. The SR expression cassette with thenopaline synthase promoter from Agrobacterium tumefaciens (Shaw et al.,Nucl. Acids Res. 12:7831-7846,1984) or modified nos promoters isdescribed below. The PAT˜GFP cassette contains a maize-optimized geneencoding phosphinothricin acetyltransferase (moPAT, see WO 98/30701)followed by a sequence encoding 4×(GSSS), a flexible polypeptide linkerof GLY-SER-SER-SER, and then a maize-optimized nucleic acid sequenceencoding Green Fluorescence Protein (GFP; see WO 98/01575). This PAT˜GFPfusion construct is driven by the maize ubiquitin promoter (Christensenet al., Plant Mol. Biol. 18:675-689, 1992) and contains a potatoproteinase inhibitor II 3′ sequence (An et al., Plant Cell1:115-122,1989).

Transgenic inbred maize plants containing a co-segregating SR expressioncassette and the UBI::PAT˜GFP expression cassette are crossed towild-type (non-transformed) inbred maize plants (using thenon-transformed parent as the pollen donor). As expected from such across, the developing embryos on these ears segregate either fortransgene expression or wild-type. Immature embryos are harvested 12days after pollination and transformed with an Agrobacterium binaryplasmid containing UBI::moCAH::pinII (moCAH is a maize optimized [forcodon usage] gene that encodes for the Myrothecium verucaria cyanamidehydratase protein[CAH] that can hydrate cyanamide to non-toxic urea). Astandard Agrobacterium-mediated transformation protocol (U. S. Pat. No.5,981,840) adapted for cyanamide selection (see WO 98/30701) is used,with additional modifications listed below. Agrobacterium is grown tolog phase in liquid minimal-A medium containing 100 μM acetosyringoneand spectinomycin. Embryos are immersed in a log phase suspension ofAgrobacterium adjusted to obtain 3×10⁸ CFU's/ml. Embryos are thenco-cultured on culture medium with acetosyringone for 3 days at 20° C.After 3 days the embryos are returned to standard culture medium with100 mg/l carbenicillin added to kill residual Agrobacterium. After anadditional 4 days the segregating embryos are divided into GFP positiveand GFP negative populations and moved to fresh culture medium with 50mg/l cyanamide for selection. After 8 weeks the numbers of transformedcolonies are determined.

Since the PAT˜GFP and SR expression cassettes are co-segregating, GFPexpression is used to separate segregating transgenic (PAT˜GFP+/SR+) andnon-transgenic (wild-type) embryos after Agrobacterium-mediatedtransformation, and then these separate populations are cultured andselected as independent groups. Using embryos from three different earsco-segregating for GFP and SR, we expect the SR-containing embryos toexhibit a much higher transformation frequency demonstrating thatectopic SR expression improves re-transformation frequencies. For earsfrom which the wild-type embryos (non-transgenic segregants) producevery low levels (or no) transformants, we expect the GFP+/SR-containingembryos from the same ears to produce cyanamide-resistant transformantsat approximately a 5-10% frequency. In ears in which the wild-type,non-transformed embryos produce higher levels of transformants (forexample, upwards of 10%), we expect the transformation frequencies fromthe SR expressing embryos to be elevated to even greater levels, i.e.upwards of 30-40%.

Particle gun transformation re-transformations. As the starting pointfor particle gun-mediated re-transformation experiments, regeneratedmaize inbred T0 transformants are produced containing maize SRexpression cassettes and UBI::moPAT˜GFP::pinII. Transformants containingUBI::moPAT˜GFP::pinII and SR expression cassettes are tested; with SRbeing driven by a nos promoter. As a control, a non-functional versionof SR is used, in which the SR coding sequence is frame-shifted by 1position after the START codon, resulting in essentially the same mRNAspecies but producing a non-functional protein. Expression of thisframe-shifted sequence (abbreviated “f-shift” below) is driven by thenos promoter. As mentioned above for the functional SR genes, thisf-shift SR cassette co-segregates with GFP in the T1 progeny embryos.

Transgenic maize inbred plants containing a co-segregating SR expressioncassette and the UBI::PAT˜GFP expression cassette are crossed towild-type (non-transformed) PHP38 plants (using the non-transformedparent as the pollen donor). As expected from such a cross, thedeveloping embryos on these ears segregate either for transgeneexpression or wild-type. Embryos co-segregating for GFP and SR(functional and frame-shift (fs) versions) are transformed using aparticle gun using the standard PHP38 immature embryo bombardmenttransformation protocol (Songstad D. D. et al., In Vitro Cell Dev. Biol.Plant 32:179-183,1996). Cells are transformed by culturing maizeimmature embryos (approximately 1-1.5 mm in length) onto 560P mediumcontaining N6 salts, Erikkson's vitamins, 0,69 g/l proline, 2 mg/l 2.4-Dand 3% sucrose. After 4-5 days of incubation in the dark at 28° C.,embryos are removed from 560P medium and cultured, scutellum up, onto560Y medium which is equivalent to 560P but contains 12% sucrose.Embryos are allowed to acclimate to this medium for 3 h prior totransformation. The scutellar surface of the immature embryos istargeted using particle bombardment with a ubi:moCAH:pinII plasmid.Embryos are transformed using the PDS-1000 Helium Gun from Bio-Rad atone shot per sample using 650 P.S.I. rupture disks. DNA delivered pershot averages at 0.1667 ug. Following bombardment, all embryos aremaintained on 560L medium (N6 salts, Eriksson's vitamins, 0.5 mg/lthiamine, 20 g/l sucrose, 1 mg/l 2,4-D, 2.88 g/l proline, 2.0 g/lgelrite, and 8.5 mg/l silver nitrate). After 2-7 days post-bombardment,all the embryos from both treatments are transferred onto N6-basedmedium containing 50mg/l cyanamide (560P medium described supra, with50mg/l cyanamide). Plates are maintained at 28° C. in the dark and areobserved for colony recovery with transfers to fresh medium occurringevery two to three weeks. Early in the sub-culture regime, GFP+ and GFP−embryos are separated. These two sub-populations are subsequentlycultured and analyzed as separate treatments. The PAT˜GFP expressioncassette and the SR expression cassette co-segregate together, and thusthe presence of GFP expression is used to separate SR+ and SR− progenyfor analysis.

Comparing PAT˜GFP+/SR+ transgenic embryos with wild-type(non-transgenic) embryos from the same ear we expect will show that theoverall recovery of cyanimide-resistant transformants is much higher forthe SR transgenic embryos. For ears from PAT˜GFP+/SRfs transgenic plants(containing the frame-shift control) we expect there to be nosignificant improvement in transformation frequencies over segregatingwild-type embryos.

EXAMPLE 14 Using SR to Improve Soybean Transformation

Delivery of the SR gene can be accomplished through numerouswell-established methods for plant cells, including for example particlebombardment, sonication, PEG treatment or electroporation ofprotoplasts, electroporation of intact tissue, silica-fiber methods,microinjection or Agrobacterium-mediated transformation. Using one ofthe above methods, DNA is introduced into soybean cells capable ofgrowth on suitable soybean culture medium. The SR gene (zmSRp30, zmSRp31or zmSRp32) is cloned into a cassette with a constitutive promoter (forexample, the SCP-1 promoter which confers constitutive expression insoybean, see PHI Patent application WO 99/43838) and a 3′ sequence suchas the nos 3′region. Particle bombardment is used to introduce theSCP1::SR::nos-containing plasmid along with a SCP1::HYG::nos-containingplasmid (which, when expressed produces a protein which confershygromycin resistance) into soybean cells capable of growth on suitablesoybean culture medium. Such competent cells can be from soybeansuspension culture, cell culture on solid medium, freshly isolatedcotyledonary nodes or meristem cells. Suspension-cultured somaticembryos of Jack, a Glycine max (I.) Merrill cultivar, are used as thetarget for co-delivery of a SR and a HYG-expressing plasmid. For targettissues receiving the SR expression cassette, transformation frequencyis improved. Media for induction of cell cultures with high somaticembryogenic morphology, for establishing suspensions, and formaintenance and regeneration of somatic embryos are described (Bailey MA, Boerma H R, Parrott W A, 1993 Genotype effects on proliferativeembryogenesis and plant regeneration of soybean, In Vitro Cell Dev Biol29P:102-108). Likewise, methods for particle-mediated transformation ofsoybean are well established in the literature, see for example StewartN C, Adang M J, All J N, Boerma H R, Cardineau G, Tucker D, Parrott W A,1996, Genetic transformation, recovery and characterization of fertilesoybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene,Plant Physiol 112:121-129.

1. An isolated nucleic acid encoding a polypeptide having RNA splicingactivity comprising: (a) a polynucleotide that encodes a polypeptide ofSEQ ID NOS: 2 or 4; (b) a polynucleotide that encodes a polypeptidehaving at least 90% identity to SEQ ID NOS: 2 or 4; (c) a polynucleotidecomprising the sequence set forth in SEQ ID NO:
 30. 2. A vectorcomprising at least one nucleic acid of claim
 1. 3. A recombinantexpression cassette comprising a nucleic acid of claim 1 operably linkedto a promoter.
 4. A host cell comprising the recombinant expressioncassette of claim 3, wherein said host cell is a plant cell or abacterial cell.
 5. The host cell of claim 4 wherein said host cell is aplant cell.
 6. The plant cell of claim 5 wherein said plant cell is acorn, soybean, sorghum, sunflower, safflower, wheat, rice, alfalfa, orBrassica cell.
 7. A transgenic plant comprising at least one expressioncassette of claim
 3. 8. A transgenic seed from the plant of claim
 7. 9.A transgenic plant produced by growing the seed of claim
 8. 10. A methodof increasing the splicing of a Geminivirus comprising introducing theisolated nucleic acid of claim 1 into a plant cell.
 11. The method ofclaim 10 wherein the virus is a wheat dwarf or maize streak virus. 12.The method of claim 10 wherein Rep A splicing is increased.
 13. Anisolated ribonucleic acid sequence encoded by a polynucleotide of claim1.